Methods and compositions using rna interference and antisense oligonucleotides for inhibition of kras

ABSTRACT

The invention relates to the inhibition of expression of mutant KRAS sequences using RNA interference, antisense oligonucleotides, and chemically-modified oligonucleotides.

STATEMENT OF PRIORITY

This application is a divisional of and claims priority to U.S.application Ser. No. 16/842,404, filed Apr. 7, 2020, now U.S. Pat. No.11,180,759, which is a continuation-in-part of U.S. application Ser. No.16/070,600, filed Jul. 17, 2018, now U.S. Pat. No. 10,619,159, which isa 35 U.S.C. § 371 national phase application of PCT ApplicationPCT/US2017/014013 filed Jan. 19, 2017, which claims the benefit of U.S.Provisional Application Ser. No. 62/280,458, filed Jan. 19, 2016, theentire contents of each of which are incorporated by reference herein inits entirety.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 5470-773IPDV_ST25.txt, 59,648 bytes in size, generatedon Nov. 18, 2021 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

The invention relates to the inhibition of expression of mutant KRASsequences using RNA interference, antisense oligonucleotides, andchemically-modified oligonucleotides.

BACKGROUND OF THE INVENTION

Since its discovery in 1982, the RAS family of genes has beencharacterized as an important class of proto-oncogenes (Cox et al., Nat.Rev. Drug Discov. 13:828 (2014)). Through three decades of extensiveresearch, mutational activation of certain RAS genes (KRAS, NRAS, andHRAS) has been implicated in nearly one-third of all cancers (Pecot etal., Mol. Cancer Ther. 13:2876 (2014)). In particular, KRAS mutationsare observed most frequently, both exclusively and in conjunction withthe other RAS isoforms (Cox et al., Nat. Rev. Drug Discov. 13:828(2014)). Yet in spite of efforts to develop inhibitors for this highlyprevalent mutation, no strong therapeutic candidates have emerged, thusearning the KRAS gene its reputation as an elusively “undruggable”target.

The RAS genes encode a family of small GTPases that act upon downstreameffector proteins to promote cell survival, growth, and proliferation(Khosravi-Far et al., Cancer Metastasis Rev. 13:67 (1994)). Properfunction of the RAS proteins relies upon activation via a guaninenucleotide exchange factor (GEF) to its active, GTP-bound form as wellas membrane association of the RAS-GTP complex, both of which have beenproposed as targets for KRAS inhibition. However, due to low efficacyand target specificity of previously proposed therapeutic agents indirectly inhibiting KRAS, current measures to target the KRAS pathwayfocus predominantly on inhibition of downstream effector proteins (Coxet al., Nat. Rev. Drug Discov. 13:828 (2014)). Nevertheless, despitechallenges in developing a small molecule to directly down-regulate geneactivity, KRAS remains a therapeutically relevant target due to itsprevalence as a driving mutation in human cancers.

Advances in RNA interference (RNAi) suggest its potential as aneffective means of knocking down KRAS expression. RNAi therapy uses theinteraction of an exogenous small interfering RNA (siRNA) and endogenousenzymatic machinery, termed an RNA-induced silencing complex (RISC), toselectively silence specific genes at the mRNA level (Pecot et al., Nat.Rev. Cancer 11:59 (2011)). A recent study has revealed the efficacy ofRNAi as a well-tolerated therapy for inducing metastatic regression inhuman cancer patients (Tabernero et al., Cancer Discov. 3:406 (2013)).In addition, using nanoliposomes we have recently verified the efficacyof siRNA delivery for knockdown of human KRAS in various lung and coloncancer models, both in vitro and in vivo (Pecot et al., Mol. CancerTher. 13:2876 (2014)).

However, there remains a lack of target-specificity for mutant KRAS overthe wild-type (WT) allele. Despite the oncogenic properties of themutant allele, WT KRAS is necessary for proper response toextra-cellular inputs that promote viability in non-cancerous cells(Khosravi-Far et al., Cancer Metastasis Rev. 13:67 (1994)). As such,there is a need for inhibitors that target mutant KRAS while sparing WTKRAS.

Accordingly, the present invention overcomes the deficiencies in the artby providing compositions and methods using RNA interference forspecific inhibition of mutant KRAS sequences.

SUMMARY OF THE INVENTION

The present invention is based on the identification of RNA moleculesthat inhibit expression of mutant KRAS sequences while sparingexpression of WT KRAS. Accordingly, one aspect of the invention relatesto a double stranded RNA molecule comprising an antisense strand and asense strand, wherein the nucleotide sequence of the antisense strand iscomplementary to a region of the nucleotide sequence of a synthetichuman KRAS gene that contains the missense mutations G12C, G12D, andG13D or the missense mutations G12C, G12V, and G13D, the regionconsisting essentially of about 18 to about 25 consecutive nucleotides;wherein the double stranded RNA molecule inhibits expression of a mutanthuman KRAS gene comprising one or more of the missense mutations G12C,G12D, G12V, and G13D and minimally inhibits expression of wild-typehuman KRAS.

Another aspect of the invention relates to a composition, e.g., apharmaceutical composition, comprising one or more of the RNA moleculesof the invention.

A further aspect of the invention relates to a method of inhibitingexpression of a mutant human KRAS gene comprising one or more of themissense mutations G12C, G12D, G12V, and G13D in a cell, the methodcomprising contacting the cell with the RNA molecule of the invention,thereby inhibiting expression of the mutant human KRAS gene in the cell.

An additional aspect of the invention relates to a method of treatingcancer in a subject in need thereof, wherein the cancer comprises amutant human KRAS gene comprising one or more of the missense mutationsG12C, G12D, G12V, and G13D, the method comprising delivering to thesubject the RNA molecule of the invention, thereby treating cancer inthe subject.

Another aspect of the invention relates to the use of the RNA moleculesof the invention to inhibit expression of a mutant human KRAS genecomprising one or more of the missense mutations G12C, G12D, G12V. andG13D in a cell and to treat cancer in a subject in need thereof, whereinthe cancer comprises a mutant human KRAS gene comprising one or more ofthe missense mutations G12C, G12D, G12V, and G13D.

A further aspect of the invention relates to an antisenseoligonucleotide targeted to a synthetic human KRAS mRNA that encodes themissense mutations G12C, G12D, and G13D, wherein the antisenseoligonucleotide is 16-25 nucleotides in length and comprises thesequence TCTTGCCTACGTCATA (SEQ ID NO:114).

An additional aspect of the invention relates to an antisenseoligonucleotide targeted to a naturally-occurring human KRAS mRNAencoding a mutation selected from G12C, G12D, G12V, and G13D, whereinthe antisense oligonucleotide is 16-25 nucleotides in length andcomprises a sequence selected from:

a) TCTTGCCTACGCCACA (SEQ ID NO:117) targeted to a human KRAS mRNAencoding a G12C mutation;

b) TCTTGCCTACGCCATC (SEQ ID NO:118) targeted to a human KRAS mRNAencoding a G12D mutation;

c) TCTTGCCTACGCCAAC (SEQ ID NO:119) targeted to a human KRAS mRNAencoding a G12V mutation:

d) TCTTGCCTACGTCACC (SEQ ID NO:120) targeted to a human KRAS mRNAencoding a G13D mutation; or

e) a sequence at least 90% identical to any one of a) to d) wherein theantisense oligonucleotide comprises at least one non-naturally occurringchemical modification.

Another aspect of the invention relates to a siRNA molecule targeted toa naturally-occurring human KRAS mRNA encoding a mutation selected fromG12C, G12D, G12V. and G13D, wherein the siRNA molecule comprises atleast one chemical modification, and wherein the siRNA moleculecomprises one of the following pairs of sequences: sense strand of SEQID NO:128 and antisense strand of SEQ ID NO:129;

-   -   sense strand of SEQ ID NO:130 and antisense strand of SEQ ID        NO:131;    -   sense strand of SEQ ID NO:132 and antisense strand of SEQ ID        NO:133;    -   sense strand of SEQ ID NO:134 and antisense strand of SEQ ID        NO:135;    -   sense strand of SEQ ID NO:136 and antisense strand of SEQ ID        NO:137;    -   sense strand of SEQ ID NO:138 and antisense strand of SEQ ID        NO:139;    -   sense strand of SEQ ID NO:140 and antisense strand of SEQ ID        NO:141;    -   sense strand of SEQ ID NO:142 and antisense strand of SEQ ID        NO:143;    -   sense strand of SEQ ID NO:144 and antisense strand of SEQ ID        NO:145;    -   sense strand of SEQ ID NO:146 and antisense strand of SEQ ID        NO:147;    -   sense strand of SEQ ID NO:148 and antisense strand of SEQ ID        NO:149;    -   sense strand of SEQ ID NO:150 and antisense strand of SEQ ID        NO:151;    -   sense strand of SEQ ID NO:152 and antisense strand of SEQ ID        NO:153;    -   sense strand of SEQ ID NO:154 and antisense strand of SEQ ID        NO:155;    -   sense strand of SEQ ID NO:156 and antisense strand of SEQ ID        NO:157; or    -   sense strand of SEQ ID NO:158 and antisense strand of SEQ ID        NO:159; or a sequence at least 90% identical thereto.

An additional aspect of the invention relates to an siRNA moleculetargeted to human KRAS mRNA, wherein the sense strand of the siRNAcomprises the sequence of SEQ ID NO:50 or SEQ ID NO:51, and wherein thesiRNA comprises at least one non-naturally occurring chemicalmodification.

Another aspect of the invention relates to a composition, e.g., apharmaceutical composition, comprising one or more of the antisenseoligonucleotides or siRNAs molecules of the invention.

A further aspect of the invention relates to a method of inhibitingexpression of a mutant human KRAS gene comprising one or more of themissense mutations G12C, G12D, G12V, and G13D in a cell, the methodcomprising contacting the cell with the antisense oligonucleotides orsiRNAs molecules of the invention, thereby inhibiting expression of themutant human KRAS gene in the cell.

An additional aspect of the invention relates to a method of treatingcancer in a subject in need thereof, wherein the cancer comprises amutant human KRAS gene comprising one or more of the missense mutationsG12C, G12D, G12V, and G13D, the method comprising delivering to thesubject the antisense oligonucleotides or siRNAs molecules of theinvention, thereby treating cancer in the subject.

Another aspect of the invention relates to the use of the antisenseoligonucleotides or siRNAs molecules of the invention to inhibitexpression of a mutant human KRAS gene comprising one or more of themissense mutations G12C, G12D, G12V, and G13D in a cell and to treatcancer in a subject in need thereof, wherein the cancer comprises amutant human KRAS gene comprising one or more of the missense mutationsG12C, G12D, G12V, and G13D.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows KRAS siRNA sequences (SEQ ID NOS:40-51). TMS siRNAsequences were designed to bind the G domain of the human KRAS gene atcodons 12 and 13 and target three point mutations (each indicated withan asterisk). Underlining indicates the remaining base pairs targeted bythe siRNA (sense). Sequences for G12C and G12D siRNAs were obtained fromFleming et al., Mol. Cancer Res. 3:413 (2005). Positive control siRNAs(Seq2 and Seq3) were obtained from Pecot et al., Mol. Cancer Ther.13:2876 (2014) and targeted a downstream coding region of the KRAS mRNA.

FIGS. 2A-2B show KRAS expression levels with mutant-specific (MS) andcontrol siRNAs. NIH 3T3 cells infected with human WT, G12C, G12D, G12V,or G13D KRAS were reverse transfected with either (A) MS siRNA sequences(12CD113D_1, 12CD113D_2, 12CD13D_3, 12CD13D_4, 12CV13D_1, and 12CV13D_2)or (B) control mutant-specific siRNA or non-specific sequences.

FIG. 3 shows the testing of custom KRAS siRNA sequences 12CD13D_1 and12CD13D_4 in a KRAS G12D mutant lung cancer cell line.

FIG. 4 shows the library of siRNA sequences (SEQ ID NOS:45-51) used fortesting all possible siRNA sequence permutations between the customsiRNA sequences.

FIG. 5 shows the relative expression of wild-type and mutant KRAS mRNAsin 3T3 cells.

FIG. 6 shows a schematic of the antisense oligonucleotide screen againstthe synthetic KRAS gene (SEQ ID NOS:52 and 53).

FIG. 7 shows the ability of 16 antisense oligonucleotides to inhibitmutant KRAS expression in A431 cells. The A431 cells have beengenetically-engineered to have the KRAS wild-type allele removed andindividual A431 clones were created to express the human KRAS mutantalleles, either KRAS G12C, KRAS G12D, KRAS G12V, or KRAS G13D (asshown); thus controlling for gymnotic delivery mechanisms,oligonucleotide trafficking and RNAse H silencing activity.

FIG. 8 shows the ability of 6 antisense oligonucleotides to inhibitmutant KRAS expression in genetically-engineered A431 cells at differentconcentrations.

FIG. 9 shows the ability of chemically-modified ASO16 antisenseoligonucleotides to inhibit mutant KRAS expression ingenetically-engineered A431 cells.

FIG. 10 shows the activity of fully modified siRNAs targeted to the KRASG12C mutation in genetically-engineered A431 cells expressing KRAS G12C.

FIG. 11 shows the activity of fully modified siRNAs targeted to the KRASG12C mutation in genetically-engineered A431 cells expressing KRAS G12C.

FIG. 12 shows the activity of fully modified siRNAs targeted to the KRASG12D mutation in genetically-engineered A431 cells expressing KRAS G12D.

FIG. 13 shows the activity of fully modified siRNAs targeted to the KRASG12D mutation in genetically-engineered A431 cells expressing KRAS G12D.

FIG. 14 shows the activity of fully modified siRNAs targeted to the KRASG12V mutation in genetically-engineered A431 cells expressing KRAS G12V.

FIG. 15 shows the activity of fully modified siRNAs targeted to the KRASG12V mutation in genetically-engineered A431 cells expressing KRAS G12V.

FIG. 16 shows the activity of fully modified siRNAs targeted to the KRASG13D mutation in genetically-engineered A431 cells expressing KRAS G13D.

FIG. 17 shows the activity of fully modified siRNAs targeted to the KRASG13D mutation in genetically-engineered A431 cells expressing KRAS G13D.

FIG. 18 shows the activity of fully modified siRNAs targeted to KRAS inHCT116 (KRAS G13D mutant) and LU65 (KRAS G12C mutant) cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in more detail withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents, patentpublications and other references cited herein are incorporated byreference in their entireties for the teachings relevant to the sentenceand/or paragraph in which the reference is presented.

Nucleotide sequences are presented herein by single strand only, in the5′ to 3′ direction, from left to right, unless specifically indicatedotherwise. Nucleotides and amino acids are represented herein in themanner recommended by the IUPAC-IUB Biochemical Nomenclature Commission,or (for amino acids) by either the one-letter code, or the three lettercode, both in accordance with 37 C.F.R. § 1.822 and established usage.

Except as otherwise indicated, standard methods known to those skilledin the art may be used for cloning genes, amplifying and detectingnucleic acids, and the like. Such techniques are known to those skilledin the art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual 2nd Ed. (Cold Spring Harbor, N.Y., 1989); Ausubel et al. CurrentProtocols in Molecular Biology (Green Publishing Associates, Inc. andJohn Wiley & Sons, Inc., New York).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination.

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted.

To illustrate, if the specification states that a complex comprisescomponents A, B and C, it is specifically intended that any of A, B orC, or a combination thereof, can be omitted and disclaimed singularly orin any combination.

Definitions

As used in the description of the invention and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

The term “about,” as used herein when referring to a measurable valuesuch as an amount of polypeptide, dose, time, temperature, enzymaticactivity or other biological activity and the like, is meant toencompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% ofthe specified amount.

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps and those that do not materially affect the basic andnovel characteristic(s) of the claimed invention. Thus, the term“consisting essentially of” as used herein should not be interpreted asequivalent to “comprising.”

The term “consists essentially of” (and grammatical variants), asapplied to a polynucleotide sequence of this invention, means apolynucleotide that consists of both the recited sequence (e.g., SEQ IDNO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)additional nucleotides on the 5′ and/or 3′ ends of the recited sequencesuch that the function of the polynucleotide is not materially altered.The total of ten or less additional nucleotides includes the totalnumber of additional nucleotides on both ends added together.

The term “materially altered,” as applied to polynucleotides of theinvention, refers to an increase or decrease in ability to inhibitexpression of a target mRNA of at least about 50% or more as compared tothe expression level of a polynucleotide consisting of the recitedsequence.

The term “enhance” or “increase” refers to an increase in the specifiedparameter of at least about 1.25-fold, 1.5-fold. 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold.

The term “inhibit” or “reduce” or grammatical variations thereof as usedherein refers to a decrease or diminishment in the specified level oractivity of at least about 15%, 25%, 35%. 40%, 50%, 60%, 75%, 80%, 90%,95% or more. In particular embodiments, the inhibition or reductionresults in little or essentially no detectible activity (at most, aninsignificant amount, e.g., less than about 10% or even 5%).

A “therapeutically effective” amount as used herein is an amount thatprovides some improvement or benefit to the subject. Alternativelystated, a “therapeutically effective” amount is an amount that willprovide some alleviation, mitigation, or decrease in at least oneclinical symptom in the subject (e.g., in the case of cancer, reductionin tumor burden, prevention of further tumor growth, prevention ofmetastasis, or increase in survival time). Those skilled in the art willappreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

By the terms “treat,” “treating,” or “treatment of.” it is intended thatthe severity of the subject's condition is reduced or at least partiallyimproved or modified and that some alleviation, mitigation or decreasein at least one clinical symptom is achieved.

“Prevent” or “preventing” or “prevention” refer to prevention or delayof the onset of the disorder and/or a decrease in the severity of thedisorder in a subject relative to the severity that would develop in theabsence of the methods of the invention. The prevention can be complete,e.g., the total absence of cancer in a subject. The prevention can alsobe partial, such that the occurrence or severity of cancer in a subjectis less than that which would have occurred without the presentinvention.

As used herein, “nucleic acid,” “nucleotide sequence,” and“polynucleotide” are used interchangeably and encompass both RNA andDNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemicallysynthesized) DNA or RNA and chimeras of RNA and DNA. The termpolynucleotide, nucleotide sequence, or nucleic acid refers to a chainof nucleotides without regard to length of the chain. The nucleic acidcan be double-stranded or single-stranded. Where single-stranded, thenucleic acid can be a sense strand or an antisense strand. The nucleicacid can be synthesized using oligonucleotide analogs or derivatives(e.g., inosine or phosphorothioate nucleotides). Such oligonucleotidescan be used, for example, to prepare nucleic acids that have alteredbase-pairing abilities or increased resistance to nucleases. The presentinvention further provides a nucleic acid that is the complement (whichcan be either a full complement or a partial complement) of a nucleicacid, nucleotide sequence, or polynucleotide of this invention. WhendsRNA is produced synthetically, less common bases, such as inosine,5-methylcytosine, 6-methyladenine, hypoxanthine and others can also beused for antisense, dsRNA, and ribozyme pairing. For example,polynucleotides that contain C-5 propyne analogues of uridine andcytidine have been shown to bind RNA with high affinity and to be potentantisense inhibitors of gene expression. Other modifications, such asmodification to the phosphodiester backbone, or the 2′-hydroxy in theribose sugar group of the RNA can also be made.

An “isolated polynucleotide” is a nucleotide sequence (e.g., DNA or RNA)that is not immediately contiguous with nucleotide sequences with whichit is immediately contiguous (one on the 5′ end and one on the 3′ end)in the naturally occurring genome of the organism from which it isderived. Thus, in one embodiment, an isolated nucleic acid includes someor all of the 5′ non-coding (e.g., promoter) sequences that areimmediately contiguous to a coding sequence. The term thereforeincludes, for example, a recombinant DNA that is incorporated into avector, into an autonomously replicating plasmid or virus, or into thegenomic DNA of a prokaryote or eukaryote, or which exists as a separatemolecule (e.g., a cDNA or a genomic DNA fragment produced by PCR orrestriction endonuclease treatment), independent of other sequences. Italso includes a recombinant DNA that is part of a hybrid nucleic acidencoding an additional polypeptide or peptide sequence. An isolatedpolynucleotide that includes a gene is not a fragment of a chromosomethat includes such gene, but rather includes the coding region andregulatory regions associated with the gene, but no additional genesnaturally found on the chromosome.

The term “isolated” can refer to a nucleic acid, nucleotide sequence orpolypeptide that is substantially free of cellular material, viralmaterial, and/or culture medium (when produced by recombinant DNAtechniques), or chemical precursors or other chemicals (when chemicallysynthesized). Moreover, an “isolated fragment” is a fragment of anucleic acid, nucleotide sequence or polypeptide that is not naturallyoccurring as a fragment and would not be found in the natural state.“Isolated” does not mean that the preparation is technically pure(homogeneous), but it is sufficiently pure to provide the polypeptide ornucleic acid in a form in which it can be used for the intended purpose.

An “isolated cell” refers to a cell that is separated from othercomponents with which it is normally associated in its natural state.For example, an isolated cell can be a cell in culture medium and/or acell in a pharmaceutically acceptable carrier of this invention. Thus,an isolated cell can be delivered to and/or introduced into a subject.In some embodiments, an isolated cell can be a cell that is removed froma subject and manipulated as described herein ex vivo and then returnedto the subject.

The term “fragment,” as applied to a polynucleotide, will be understoodto mean a nucleotide sequence of reduced length relative to a referencenucleic acid or nucleotide sequence and comprising, consistingessentially of, and/or consisting of a nucleotide sequence of contiguousnucleotides identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99%identical) to the reference nucleic acid or nucleotide sequence. Such anucleic acid fragment according to the invention may be, whereappropriate, included in a larger polynucleotide of which it is aconstituent. In some embodiments, such fragments can comprise, consistessentially of, and/or consist of oligonucleotides having a length of atleast about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, or more consecutive nucleotides of a nucleic acid or nucleotidesequence according to the invention.

The term “fragment,” as applied to a polypeptide, will be understood tomean an amino acid sequence of reduced length relative to a referencepolypeptide or amino acid sequence and comprising, consistingessentially of, and/or consisting of an amino acid sequence ofcontiguous amino acids identical or almost identical (e.g., 90%, 92%,95%, 98%, 99% identical) to the reference polypeptide or amino acidsequence. Such a polypeptide fragment according to the invention may be,where appropriate, included in a larger polypeptide of which it is aconstituent. In some embodiments, such fragments can comprise, consistessentially of, and/or consist of peptides having a length of at leastabout 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, or more consecutive amino acids of a polypeptide or amino acidsequence according to the invention.

A “vector” is any nucleic acid molecule for the cloning of and/ortransfer of a nucleic acid into a cell. A vector may be a replicon towhich another nucleotide sequence may be attached to allow forreplication of the attached nucleotide sequence. A “replicon” can be anygenetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome)that functions as an autonomous unit of nucleic acid replication invivo, i.e., capable of replication under its own control. The term“vector” includes both viral and nonviral (e.g., plasmid) nucleic acidmolecules for introducing a nucleic acid into a cell in vitro, ex vivo,and/or in vivo. A large number of vectors known in the art may be usedto manipulate nucleic acids, incorporate response elements and promotersinto genes, etc. For example, the insertion of the nucleic acidfragments corresponding to response elements and promoters into asuitable vector can be accomplished by ligating the appropriate nucleicacid fragments into a chosen vector that has complementary cohesivetermini. Alternatively, the ends of the nucleic acid molecules may beenzymatically modified or any site may be produced by ligatingnucleotide sequences (linkers) to the nucleic acid termini. Such vectorsmay be engineered to contain sequences encoding selectable markers thatprovide for the selection of cells that contain the vector and/or haveincorporated the nucleic acid of the vector into the cellular genome.Such markers allow identification and/or selection of host cells thatincorporate and express the proteins encoded by the marker. A“recombinant” vector refers to a viral or non-viral vector thatcomprises one or more heterologous nucleotide sequences (i.e.,transgenes), e.g., two, three, four, five or more heterologousnucleotide sequences.

Viral vectors have been used in a wide variety of gene deliveryapplications in cells, as well as living animal subjects. Viral vectorsthat can be used include, but are not limited to, retrovirus,lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus,vaccinia virus, herpes virus, Epstein-Barr virus, and/or adenovirusvectors. Non-viral vectors include, but are not limited to, plasmids,liposomes, electrically charged lipids (cytofectins), nucleicacid-protein complexes, and biopolymers. In addition to a nucleic acidof interest, a vector may also comprise one or more regulatory regions,and/or selectable markers useful in selecting, measuring, and monitoringnucleic acid transfer results (delivery to specific tissues, duration ofexpression, etc.).

Vectors may be introduced into the desired cells by methods known in theart, e.g., transfection, electroporation, microinjection, transduction,cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a nucleic acid vectortransporter (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu etal., J. Biol. Chem. 263:14621 (1988); and Hartmut et al., CanadianPatent Application No. 2,012,311, filed Mar. 15, 1990).

In some embodiments, a polynucleotide of this invention can be deliveredto a cell in vivo by lipofection. Synthetic cationic lipids designed tolimit the difficulties and dangers encountered with liposome-mediatedtransfection can be used to prepare liposomes for in vivo transfectionof a nucleotide sequence of this invention (Feigner et al., Proc. Natl.Acad. Sci. USA 84:7413 (1987); Mackey, et al., Proc. Natl. Acad. Sc.U.S.A. 85:8027 (1988); and Ulmer et al., Science 259:1745 (1993)). Theuse of cationic lipids may promote encapsulation of negatively chargednucleic acids, and also promote fusion with negatively charged cellmembranes (Feigner et al., Science 337:387 (1989)). Particularly usefullipid compounds and compositions for transfer of nucleic acids aredescribed in International Patent Publications WO95/18863 andWO96/17823, and in U.S. Pat. No. 5,459,127. The use of lipofection tointroduce exogenous nucleotide sequences into specific organs in vhvohas certain practical advantages. Molecular targeting of liposomes tospecific cells represents one area of benefit. It is clear thatdirecting transfection to particular cell types would be particularlypreferred in a tissue with cellular heterogeneity, such as pancreas,liver, kidney, and the brain. Lipids may be chemically coupled to othermolecules for the purpose of targeting (Mackey, et al., 1988, supra).Targeted peptides, e.g., hormones or neurotransmitters, and proteinssuch as antibodies, or non-peptide molecules can be coupled to liposomeschemically.

In various embodiments, other molecules can be used for facilitatingdelivery of a nucleic acid in vivo, such as a cationic oligopeptide(e.g., WO95/21931), peptides derived from nucleic acid binding proteins(e.g., WO96/25508), and/or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as naked nucleic acid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated nucleic acid delivery approaches can also be used(Curiel el al., Hum. Gene Ther. 3:147 (1992); Wu et al., J. Biol. Chem.262:4429 (1987)).

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably and encompass both peptides and proteins, unlessindicated otherwise.

A “fusion protein” is a polypeptide produced when two heterologousnucleotide sequences or fragments thereof coding for two (or more)different polypeptides not found fused together in nature are fusedtogether in the correct translational reading frame. Illustrative fusionpolypeptides include fusions of a polypeptide of the invention (or afragment thereof) to all or a portion of glutathione-S-transferase,maltose-binding protein, or a reporter protein (e.g., Green FluorescentProtein, P-glucuronidase, pi-galactosidase, luciferase, etc.),hemagglutinin, c-myc, FLAG epitope, etc.

By the term “express” or “expression” of a polynucleotide codingsequence, it is meant that the sequence is transcribed, and optionally,translated. Typically, according to the present invention, expression ofa coding sequence of the invention will result in production of thepolypeptide of the invention. The entire expressed polypeptide orfragment can also function in intact cells without purification.

As used herein, the term “gene” refers to a nucleic acid moleculecapable of being used to produce mRNA, antisense RNA, miRNA, and thelike. Genes may or may not be capable of being used to produce afunctional protein. Genes can include both coding and non-coding regions(e.g., introns, regulatory elements, promoters, enhancers, terminationsequences and 5′ and 3′ untranslated regions). A gene may be “isolated”by which is meant a nucleic acid that is substantially or essentiallyfree from components normally found in association with the nucleic acidin its natural state. Such components include other cellular material,culture medium from recombinant production, and/or various chemicalsused in chemically synthesizing the nucleic acid.

As used herein, “complementary” polynucleotides are those that arecapable of base pairing according to the standard Watson-Crickcomplementarity rules. Specifically, purines will base pair withpyrimidines to form a combination of guanine paired with cytosine (G:C)and adenine paired with either thymine (A:T) in the case of DNA, oradenine paired with uracil (A:U) in the case of RNA. For example, thesequence “A-G-T” binds to the complementary sequence “T-C-A.” It isunderstood that two polynucleotides may hybridize to each other even ifthey are not completely complementary to each other, provided that eachhas at least one region that is substantially complementary to theother.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. Complementarity between twosingle-stranded molecules may be “partial,” in which only some of thenucleotides bind, or it may be complete when total complementarityexists between the single stranded molecules. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands.

As used herein, the terms “substantially complementary” or “partiallycomplementary” mean that two nucleic acid sequences are complementary atleast about 50%, 60%, 70%, 80% or 90% of their nucleotides. In someembodiments, the two nucleic acid sequences can be complementary atleast at 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of their nucleotides.The terms “substantially complementary” and “partially complementary”can also mean that two nucleic acid sequences can hybridize under highstringency conditions and such conditions are well known in the art.

As used herein, “heterologous” refers to a nucleic acid sequence thateither originates from another species or is from the same species ororganism but is modified from either its original form or the formprimarily expressed in the cell. Thus, a nucleotide sequence derivedfrom an organism or species different from that of the cell into whichthe nucleotide sequence is introduced, is heterologous with respect tothat cell and the cell's descendants. In addition, a heterologousnucleotide sequence includes a nucleotide sequence derived from andinserted into the same natural, original cell type, but which is presentin a non-natural state, e.g., a different copy number, and/or under thecontrol of different regulatory sequences than that found in nature.

As used herein, the terms “contacting,” “introducing” and“administering” are used interchangeably, and refer to a process bywhich dsRNA of the present invention or a nucleic acid molecule encodinga dsRNA of this invention is delivered to a cell, in order to inhibit oralter or modify expression of a target gene. The dsRNA may beadministered in a number of ways, including, but not limited to, directintroduction into a cell (i.e., intracellularly) and/or extracellularintroduction into a cavity, interstitial space, or into the circulationof the organism.

“Introducing” in the context of a cell or organism means presenting thenucleic acid molecule to the organism and/or cell in such a manner thatthe nucleic acid molecule gains access to the interior of a cell. Wheremore than one nucleic acid molecule is to be introduced these nucleicacid molecules can be assembled as part of a single polynucleotide ornucleic acid construct, or as separate polynucleotide or nucleic acidconstructs, and can be located on the same or different nucleic acidconstructs. Accordingly, these polynucleotides can be introduced intocells in a single transformation event or in separate transformationevents. Thus, the term “transformation” as used herein refers to theintroduction of a heterologous nucleic acid into a cell. Transformationof a cell may be stable or transient.

“Transient transformation” in the context of a polynucleotide means thata polynucleotide is introduced into the cell and does not integrate intothe genome of the cell.

By “stably introducing” or “stably introduced” in the context of apolynucleotide introduced into a cell, it is intended that theintroduced polynucleotide is stably incorporated into the genome of thecell, and thus the cell is stably transformed with the polynucleotide.

“Stable transformation” or “stably transformed” as used herein meansthat a nucleic acid molecule is introduced into a cell and integratesinto the genome of the cell. As such, the integrated nucleic acidmolecule is capable of being inherited by the progeny thereof, moreparticularly, by the progeny of multiple successive generations.“Genome” as used herein includes the nuclear and mitochondrial genome,and therefore includes integration of the nucleic acid into, forexample, the mitochondrial genome. Stable transformation as used hereincan also refer to a transgene that is maintained extrachromasomally, forexample, as a minichromosome.

Transient transformation may be detected by, for example, anenzyme-linked immunosorbent assay (ELISA) or Western blot, which candetect the presence of a peptide or polypeptide encoded by one or moretransgene introduced into an organism. Stable transformation of a cellcan be detected by, for example, a Southern blot hybridization assay ofgenomic DNA of the cell with nucleic acid sequences which specificallyhybridize with a nucleotide sequence of a transgene introduced into anorganism. Stable transformation of a cell can be detected by, forexample, a Northern blot hybridization assay of RNA of the cell withnucleic acid sequences which specifically hybridize with a nucleotidesequence of a transgene introduced into an organism. Stabletransformation of a cell can also be detected by, e.g., a polymerasechain reaction (PCR) or other amplification reactions as are well knownin the art, employing specific primer sequences that hybridize withtarget sequence(s) of a transgene, resulting in amplification of thetransgene sequence, which can be detected according to standard methodsTransformation can also be detected by direct sequencing and/orhybridization protocols well known in the art.

Embodiments of the invention are directed to expression cassettesdesigned to express the nucleic acids of the present invention. As usedherein, “expression cassette” means a nucleic acid molecule having atleast a control sequence operably linked to a nucleotide sequence ofinterest. In this manner, for example, promoters in operable interactionwith the nucleotide sequences for the siRNAs of the invention areprovided in expression cassettes for expression in an organism or cell.

As used herein, the term “promoter” refers to a region of a nucleotidesequence that incorporates the necessary signals for the efficientexpression of a coding sequence. This may include sequences to which anRNA polymerase binds, but is not limited to such sequences and caninclude regions to which other regulatory proteins bind together withregions involved in the control of protein translation and can alsoinclude coding sequences.

Furthermore, a “promoter” of this invention is a promoter capable ofinitiating transcription in a cell of an organism. Such promotersinclude those that drive expression of a nucleotide sequenceconstitutively, those that drive expression when induced, and those thatdrive expression in a tissue- or developmentally-specific manner, asthese various types of promoters are known in the art.

For purposes of the invention, the regulatory regions (i.e., promoters,transcriptional regulatory regions, and translational terminationregions) can be native/analogous to the organism or cell and/or theregulatory regions can be native/analogous to the other regulatoryregions. Alternatively, the regulatory regions may be heterologous tothe organism or cell and/or to each other (i.e., the regulatoryregions). Thus, for example, a promoter can be heterologous when it isoperably linked to a polynucleotide from a species different from thespecies from which the polynucleotide was derived. Alternatively, apromoter can also be heterologous to a selected nucleotide sequence ifthe promoter is from the same/analogous species from which thepolynucleotide is derived, but one or both (i.e., promoter andpolynucleotide) are substantially modified from their original formand/or genomic locus, or the promoter is not the native promoter for theoperably linked polynucleotide.

The choice of promoters to be used depends upon several factors,including, but not limited to, cell- or tissue-specific expression,desired expression level, efficiency, inducibility and selectability.For example, where expression in a specific tissue or organ is desired,a tissue-specific promoter can be used. In contrast, where expression inresponse to a stimulus is desired, an inducible promoter can be used.Where continuous expression is desired throughout the cells of anorganism, a constitutive promoter can be used. It is a routine matterfor one of skill in the art to modulate the expression of a nucleotidesequence by appropriately selecting and positioning promoters and otherregulatory regions relative to that sequence.

In addition to the promoters described above, the expression cassettealso can include other regulatory sequences. As used herein, “regulatorysequences” means nucleotide sequences located upstream (5′ non-codingsequences), within or downstream (3 non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences include, but are not limited to, enhancers, introns,translation leader sequences and polyadenylation signal sequences.

The expression cassette also can optionally include a transcriptionaland/or translational termination region (i.e., termination region) thatis functional in the organism. A variety of transcriptional terminatorsare available for use in expression cassettes and are responsible forthe termination of transcription beyond the transgene and correct mRNApolyadenylation. The termination region may be native to thetranscriptional initiation region, may be native to the operably linkednucleotide sequence of interest, may be native to the host, or may bederived from another source (i.e., foreign or heterologous to thepromoter, the nucleotide sequence of interest, the host, or anycombination thereof).

A signal sequence can be operably linked to nucleic acids of the presentinvention to direct the nucleotide sequence into a cellular compartment.In this manner, the expression cassette will comprise a nucleotidesequence encoding the siRNA operably linked to a nucleic acid sequencefor the signal sequence. The signal sequence may be operably linked atthe N- or C- terminus of the siRNA.

Regardless of the type of regulatory sequence(s) used, they can beoperably linked to the nucleotide sequence of the siRNA. As used herein,“operably linked” means that elements of a nucleic acid construct suchas an expression cassette are configured so as to perform their usualfunction. Thus, regulatory or control sequences (e.g., promoters)operably linked to a nucleotide sequence of interest are capable ofeffecting expression of the nucleotide sequence of interest. The controlsequences need not be contiguous with the nucleotide sequence ofinterest, so long as they function to direct the expression thereof.

Thus, for example, intervening untranslated, yet transcribed, sequencescan be present between a promoter and a coding sequence, and thepromoter sequence can still be considered “operably linked” to thecoding sequence. A nucleotide sequence of the present invention (i.e., asiRNA) can be operably linked to a regulatory sequence, thereby allowingits expression in a cell and/or subject.

The expression cassette also can include a nucleotide sequence for aselectable marker, which can be used to select a transformed organism orcell. As used herein, “selectable marker” means a nucleic acid that whenexpressed imparts a distinct phenotype to the organism or cellexpressing the marker and thus allows such transformed organisms orcells to be distinguished from those that do not have the marker. Such anucleic acid may encode either a selectable or screenable marker,depending on whether the marker confers a trait that can be selected forby chemical means, such as by using a selective agent (e.g., anantibiotic or the like), or on whether the marker is simply a trait thatone can identify through observation or testing, such as by screening.Of course, many examples of suitable selectable markers are known in theart and can be used in the expression cassettes described herein.

In some embodiments of the present invention, the expression cassettecan comprise an expression control sequence operatively linked to anucleotide sequence that is a template for one or both strands of thedsRNA. In further embodiments, a promoter can flank either end of thetemplate nucleotide sequence, wherein the promoters drive expression ofeach individual DNA strand, thereby generating two complementary (orsubstantially complementary) RNAs that hybridize and form the dsRNA. Inalternative embodiments, the nucleotide sequence is transcribed intoboth strands of the dsRNA on one transcription unit, wherein the sensestrand is transcribed from the 5′ end of the transcription unit and theantisense strand is transcribed from the 3′ end, wherein the two strandsare separated by about 3 to about 500 basepairs, and wherein aftertranscription, the RNA transcript folds on itself to form a shorthairpin RNA (shRNA) molecule.

As used herein “sequence identity” refers to the extent to which twooptimally aligned polynucleotide or polypeptide sequences are invariantthroughout a window of alignment of components, e.g., nucleotides oramino acids. “Identity” can be readily calculated by known methodsincluding, but not limited to, those described in: ComputationalMolecular Biology (Lesk A. M., ed.) Oxford University Press. New York(1988); Biocomputing: Informatics and Genome Projects (Smith. D. W.,ed.) Academic Press, New York (1993); Computer Analysis of SequenceData, Part 1 (Griffin, A. M., and Griffin, H. G., eds.) Humana Press,New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje,G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov,M. and Devereux, J., eds.) Stockton Press, New York (1991).

As used herein, the term “substantially identical” or “corresponding to”means that two nucleic acid sequences have at least 60%, 70%, 80% or 90%sequence identity. In some embodiments, the two nucleic acid sequencescan have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of sequenceidentity.

An “identity fraction” for aligned segments of a test sequence and areference sequence is the number of identical components which areshared by the two aligned sequences divided by the total number ofcomponents in reference sequence segment, i.e., the entire referencesequence or a smaller defined part of the reference sequence.

As used herein, the term “percent sequence identity” or “percentidentity” refers to the percentage of identical nucleotides in a linearpolynucleotide sequence of a reference (“query”) polynucleotide molecule(or its complementary strand) as compared to a test (“subject”)polynucleotide molecule (or its complementary strand) when the twosequences are optimally aligned (with appropriate nucleotide insertions,deletions, or gaps totaling less than 20 percent of the referencesequence over the window of comparison). In some embodiments, “percentidentity” can refer to the percentage of identical amino acids in anamino acid sequence.

Optimal alignment of sequences for aligning a comparison window are wellknown to those skilled in the art and may be conducted by tools such asthe local homology algorithm of Smith and Waterman, the homologyalignment algorithm of Needleman and Wunsch, the search for similaritymethod of Pearson and Lipman, and optionally by computerizedimplementations of these algorithms such as GAP, BESTFIT, FASTA, andTFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc.,Burlington, Mass.). Percent sequence identity is represented as theidentity fraction multiplied by 100.

The comparison of one or more polynucleotide sequences may be to afull-length polynucleotide sequence or a portion thereof, or to a longerpolynucleotide sequence. For purposes of this invention “percentidentity” may also be determined using BLASTX version 2.0 for translatednucleotide sequences and BLASTN version 2.0 for polynucleotidesequences.

The percent of sequence identity can be determined using the “Best Fit”or “Gap” program of the Sequence Analysis Software Package™ (Version 10:Genetics Computer Group, Inc., Madison, Wis.). “Gap” utilizes thealgorithm of Needleman and Wunsch (Needleman and Wunsch, J Mol. Biol.48:443-453, 1970) to find the alignment of two sequences that maximizesthe number of matches and minimizes the number of gaps. “BestFit”performs an optimal alignment of the best segment of similarity betweentwo sequences and inserts gaps to maximize the number of matches usingthe local homology algorithm of Smith and Waterman (Smith and Waterman,Adv. Appl. Math., 2:482-489, 1981, Smith et al., Nucleic Acids Res.11:2205-2220, 1983).

Useful methods for determining sequence identity are also disclosed inGuide to Huge Computers (Martin J. Bishop, ed., Academic Press, SanDiego (1994)), and Carillo, H., and Lipton, D., (Applied Math48:1073(1988)). More particularly, preferred computer programs fordetermining sequence identity include but are not limited to the BasicLocal Alignment Search Tool (BLAST) programs which are publiclyavailable from National Center Biotechnology Information (NCBI) at theNational Library of Medicine, National Institute of Health, Bethesda,Md. 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; (Altschulet al., J. Mol. Biol. 215:403-410 (1990)); version 2.0 or higher ofBLAST programs allows the introduction of gaps (deletions andinsertions) into alignments; for peptide sequence BLASTX can be used todetermine sequence identity; and, for polynucleotide sequence BLASTN canbe used to determine sequence identity.

As used herein, “RNAi” or “RNA interference” refers to the process ofsequence-specific post-transcriptional gene silencing, mediated bydouble-stranded RNA (dsRNA). As used herein, “dsRNA” refers to RNA thatis partially or completely double stranded. Double stranded RNA is alsoreferred to as small interfering RNA (siRNA), small interfering nucleicacid (siNA), microRNA (miRNA), and the like. In the RNAi process, dsRNAcomprising a first (antisense) strand that is complementary to a portionof a target gene and a second (sense) strand that is fully or partiallycomplementary to the first antisense strand is introduced into anorganism. After introduction into the organism, the target gene-specificdsRNA is processed into relatively small fragments (siRNAs) and cansubsequently become distributed throughout the organism, leading to aloss-of-function mutation having a phenotype that, over the period of ageneration, may come to closely resemble the phenotype arising from acomplete or partial deletion of the target gene.

MicroRNAs (miRNAs) are non-protein coding RNAs, generally of betweenabout 18 to about 25 nucleotides in length. These miRNAs direct cleavagein trans of target transcripts, negatively regulating the expression ofgenes involved in various regulation and development pathways (Bartel,Cell, 116:281-297 (2004); Zhang et al. Dev. Biol. 289:3-16 (2006)). Assuch, miRNAs have been shown to be involved in different aspects ofgrowth and development as well as in signal transduction and proteindegradation. Since the first miRNAs were discovered in plants (Reinhartet al. Genes Dev 16:1616-1626 (2002), Park et al. Curr. Biol.12:1484-1495 (2002)) many hundreds have been identified. Many microRNAgenes (MIR genes) have been identified and made publicly available in adatabase (miRBase; microrna.sanger.ac.uk/sequences), miRNAs are alsodescribed in U.S. Patent Publications 2005/0120415 and 2005/144669A1,the entire contents of which are incorporated by reference herein.

Genes encoding miRNAs yield primary miRNAs (termed a “pri-miRNA”) of 70to 300 bp in length that can form imperfect stem-loop structures. Asingle pri-miRNA may contain from one to several miRNA precursors. Inanimals, pri-miRNAs are processed in the nucleus into shorter hairpinRNAs of about 65 nt (pre-miRNAs) by the RNaseIII enzyme Drosha and itscofactor DGCR8/Pasha. The pre-miRNA is then exported to the cytoplasm,where it is further processed by another RNaseIII enzyme, Dicer,releasing a miRNA/miRNA* duplex of about 22 nt in size. Many reviews onmicroRNA biogenesis and function are available, for example, see, BartelCell 116:281-297 (2004), Murchison et al. Curr. Opin. Cell Biol.16:223-229 (2004), Dugas et al. Curr. Opin. Plant Biol. 7:512-520 (2004)and Kim Nature Rev. Mol. Cell Biol. 6:376-385 (2005).

RNA Molecules

The present invention is based on the identification of RNA moleculesthat inhibit expression of mutant KRAS sequences while sparingexpression of WT KRAS. Accordingly, one aspect of the invention relatesto a double stranded RNA molecule comprising an antisense strand and asense strand, wherein the nucleotide sequence of the antisense strand iscomplementary to a region of the nucleotide sequence of a synthetichuman KRAS gene that contains the missense mutations G12C, G12D, andG13D or the missense mutations G12C, G12V, and G13D, the regionconsisting essentially of about 18 to about 25 consecutive nucleotides;wherein the double stranded RNA molecule inhibits expression of a mutanthuman KRAS gene comprising one or more of the missense mutations G12C.G12D, G12V, and G13D and minimally inhibits expression of wild-typehuman KRAS. The region of the KRAS gene targeted by the RNA moleculecomprises the nucleotides encoding residues 12 and 13. The RNA moleculesprovide decreased expression of mutant KRAS in a cell as compared to awild-type variety of the cell (e.g., a control cell or nontransformedcell). In some embodiments, expression of mutant KRAS is inhibited by atleast about 50%, e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, ormore.

A human KRAS gene containing the missense mutations G12C, G12D, and G13Dor the missense mutations G12C, G12V, and G13D does not exist in nature.Examples of a region of such artificial gene sequences include SEQ IDNOS:37 and 38, with the mutations relative to the corresponding WT KRASsequence (SEQ ID NO:39) underlined.

SEQ ID NO: 37 ACTGAATATAAACTTGTGGTAGTTGGAGCTTATGACGTAGGCAAGAGTGCCTTGACGATACAG SEQ ID NO: 38ACTGAATATAAACTTGTGGTAGTTGGAGCTTTTGACGTAGGCAAGAGTGC CTTGACGATACAGSEQ ID NO: 39 ACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAG

The double stranded RNA molecule can comprise, consist essentially of,or consist of about 18 to about 25 nucleotides (e.g., 18, 19, 20, 21,22, 23, 24, or 25 or any range therein). Additional nucleotides can beadded at the 3′ end, the 5′ end or both the 3′ and 5′ ends to facilitatemanipulation of the RNA molecule but that do not materially affect thebasic characteristics or function of the double stranded RNA molecule inRNA interference (RNAi). Additionally, one or two nucleotides can bedeleted from one or both ends of any of the sequences disclosed hereinthat do not materially affect the basic characteristics or function ofthe double stranded RNA molecule in RNAi. The term “materially affect”as used herein refers to a change in the ability to inhibit expressionof the protein encoded by the mRNA (e.g., WT KRAS) by no more than about50%, e.g., no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, or less. Such additional nucleotides can be nucleotides that extendthe complementarity of the antisense strand along the target sequenceand/or such nucleotides can be nucleotides that facilitate manipulationof the RNA molecule or a nucleic acid molecule encoding the RNAmolecule, as would be known to one of skill in the art. For example, aTT overhang at the 3′ end may be present, which is used to stabilize thesiRNA duplex and does not affect the specificity of the siRNA.

The dsRNA of the invention may optionally comprise a single strandedoverhang at either or both ends. The double-stranded structure may beformed by a single self-complementary RNA strand (i.e., forming ahairpin loop) or two complementary RNA strands. RNA duplex formation maybe initiated either inside or outside the cell. When the dsRNA of theinvention forms a hairpin loop, it may optionally comprise an intronand/or a nucleotide spacer, which is a stretch of nucleotides betweenthe complementary RNA strands, to stabilize the hairpin sequence incells. The RNA may be introduced in an amount that allows delivery of atleast one copy per cell. Higher doses of double-stranded material mayyield more effective inhibition.

In particular embodiments, the present invention provides doublestranded RNA containing a nucleotide sequence that is fullycomplementary to a region of the target gene for inhibition. However, itis to be understood that 100% complementarity between the antisensestrand of the double stranded RNA molecule and the target sequence isnot required to practice the present invention. Thus, sequencevariations that might be expected due to genetic mutation, strainpolymorphism, or evolutionary divergence can be tolerated. RNA sequenceswith insertions, deletions, and single point mutations relative to thetarget sequence may also be effective for inhibition.

In certain embodiments, the nucleotide sequence of the antisense strandcontains at least 3 mismatches with the nucleotide sequence of wild-typehuman KRAS such that the RNA molecule does not target WT KRAS and onlyminimally inhibits expression of WT KRAS. As used herein, “minimallyinhibits expression” means that expression of the protein encoded by themRNA (e.g., WT KRAS) is inhibited by no more than about 50%, e.g., nomore than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less.

In certain embodiments, the nucleotide sequence of the antisense strandcontains no more than 2 mismatches with the nucleotide sequence of amutant human KRAS gene comprising one or more of the missense mutationsG12C, G12D, G12V, and G13D. In some embodiments, the nucleotide sequenceof the antisense strand contains at least 3 mismatches with thenucleotide sequence of wild-type human KRAS and contains no more than 2mismatches with the nucleotide sequence of a mutant human KRAS genecomprising one or more of the missense mutations G12C. G12D, G12V, andG3D.

In some embodiments, the nucleotide sequence of the sense strandcomprises a nucleotide sequence that is at least about 80% identical tothe nucleotide sequence of any of SEQ ID NOS:1-9, e.g., at least about80%, 85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or moreidentical to the nucleotide sequence of any of SEQ ID NOS:1-9.

In some embodiments, the nucleotide sequence of the sense strandcomprises, consists essentially of, or consist of the nucleotidesequence of any of SEQ ID NOS:1-9.

SEQ ID NO: 1 GAGCUUAUGACGUAGGCAA SEQ ID NO: 2 AGUUGGAGCUUAUGACGUASEQ ID NO: 3 GGUAGUUGGAGCUUAUGAC SEQ ID NO: 4 GUAGUUGGAGCUUAUGACGSEQ ID NO: 5 UAGUUGGAGCUUAUGACGU SEQ ID NO: 6 GUUGGAGCUUAUGACGUAGSEQ ID NO: 7 UUGGAGCUUAUGACGUAGG SEQ ID NO: 8 UGGAGCUUAUGACGUAGGCSEQ ID NO: 9 GGAGCUUAUGACGUAGGCA

In some embodiments, the nucleotide sequence of the antisense strandcomprises a nucleotide sequence that is at least about 80% identical tothe nucleotide sequence of any of SEQ ID NOS:19-27, e.g., at least about80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the nucleotide sequence of any of SEQ ID NOS: 19-27. Insome embodiments, the nucleotide sequence of the antisense strandcomprises, consists essentially of, or consist of the nucleotidesequence of any of SEQ ID NOS: 19-27.

SEQ ID NO: 19 UUGCCUACGUCAUAAGCUC SEQ ID NO: 20 UACGUCAUAAGCUCCAACUSEQ ID NO: 21 GUCAUAAGCUCCAACUACC SEQ ID NO: 22 CGUCAUAAGCUCCAACUACSEQ ID NO: 23 ACGUCAUAAGCUCCAACUA SEQ ID NO: 24 CUACGUCAUAAGCUCCAACSEQ ID NO: 25 CCUACGUCAUAAGCUCCAA SEQ ID NO: 26 GCCUACGUCAUAAGCUCCASEQ ID NO: 27 UGCCUACGUCAUAAGCUCC

In some embodiments, one or both of the sense strand and the antisensestrand comprises a TT overhang at the 3′ end. Thus, in some embodiments,the sense strand comprises a nucleotide sequence that is at least about80% identical to the nucleotide sequence of any of SEQ ID NOS:10-18,e.g., at least about 80%, 85%, 900%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more identical to the nucleotide sequence of any of SEQ IDNOS: 10-18. In some embodiments, the nucleotide sequence of the sensestrand comprises, consists essentially of, or consist of the nucleotidesequence of any of SEQ ID NOS: 10-18.

SEQ ID NO: 10 GAGCUUAUGACGUAGGCAAdTdT SEQ ID NO: 11AGUUGGAGCUUAUGACGUAdTdT SEQ ID NO: 12 GGUAGUUGGAGCUUAUGACdTdTSEQ ID NO: 13 GUAGUUGGAGCUUAUGACGdTdT SEQ ID NO: 14UAGUUGGAGCUUAUGACGUdTdT SEQ ID NO: 15 GULTGGAGCUUAUGACGUAGdTdTSEQ ID NO: 16 UUGGAGCUUAUGACGUAGGdTdT SEQ ID NO: 17UGGAGCUUAUGACGUAGGCdTdT SEQ ID NO: 18 GGAGCUUAUGACGUAGGCAdTdT

In some embodiments, the nucleotide sequence of the antisense strandcomprise a nucleotide sequence that is at least about 80% identical tothe nucleotide sequence of any of SEQ ID NOS:28-36. e.g., at least about80%, 85%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, or moreidentical to the nucleotide sequence of any of SEQ ID NOS: 28-36. Insome embodiments, the nucleotide sequence of the antisense strandcomprises, consists essentially of, or consist of the nucleotidesequence of any of SEQ ID NOS: 28-36.

SEQ ID NO: 28 UUGCCUACGUCAUAAGCUCdTdT SEQ ID NO: 29UACGUCAUAAGCUCCAACUdTdT SEQ ID NO: 30 GUCAUAAGCUCCAACUACCdTdTSEQ ID NO: 31 CGUCAUAAGCUCCAACUACdTdT SEQ ID NO: 32ACGUCAUAAGCUCCAACUAdTdT SEQ ID NO: 33 CUACGUCAUAAGCUCCAACdTdTSEQ ID NO: 34 CCUACGUCAUAAGCUCCAAdTdT SEQ ID NO: 35GCCUACGUCAUAAGCUCCAdTdT SEQ ID NO: 36 UGCCUACGUCAUAAGCUCCdTdT

In some embodiments of this invention, the sense strand of the doublestranded RNA molecule can be fully complementary to the antisense strandor the sense strand can be substantially complementary or partiallycomplementary to the antisense strand. By substantially or partiallycomplementary is meant that the sense strand and the antisense strandcan be mismatched at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotidepairings. Such mismatches can be introduced into the sense strandsequence, e.g., near the 3′ end, to enhance processing of the doublestranded RNA molecule by Dicer, to duplicate a pattern of mismatches ina siRNA molecule inserted into a chimeric nucleic acid molecule orartificial microRNA precursor molecule of this invention, and the like,as would be known to one of skill in the art. Such modification willweaken the base pairing at one end of the duplex and generate strandasymmetry, therefore enhancing the chance of the antisense strand,instead of the sense strand, being processed and silencing the intendedgene (Geng and Ding “Double-mismatched siRNAs enhance selective genesilencing of a mutant ALS-causing Allele1” Acta Pharmacol. Sin.29:211-216 (2008); Schwarz et al. “Asymmetry in the assembly of the RNAienzyme complex” Cell 115:199-208 (2003)).

The double stranded RNA molecule of the invention may be in the form ofany type of RNA interference molecule known in the art. In someembodiments, the double stranded RNA molecule is a small interfering RNA(siRNA) molecule. In other embodiments, the double stranded RNA moleculeis a short hairpin RNA (shRNA) molecule. In other embodiments, thedouble stranded RNA molecule is part of a microRNA precursor molecule.

Antisense Oligonucleotides

One aspect of the invention relates to an antisense oligonucleotide(ASO) targeted to a synthetic human KRAS mRNA that encodes the missensemutations G12C, G12D. and G13D, wherein the ASO is 16-25 nucleotides inlength and comprises or consists essentially of the sequenceTCTTGCCTACGTCATA (SEQ ID NO:114). In some embodiments, the ASO is 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length or any rangetherein. In some embodiments, the ASO is 20,21, or 22 nucleotides inlength. In some embodiments, the ASO is 20 nucleotides in length. Incertain embodiments, at least 80% of the unspecified nucleotides in theASO are complementary to a wild-type human KRAS gene, e.g., at least80%, 85%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Incertain embodiments, at least 80% of the unspecified nucleotides in theASO are complementary to a mutant human KRAS gene, e.g., at least 80%.85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In certainembodiments, the ASO is 17-25 nucleotides in length and comprises orconsists essentially of the sequence CTCTTGCCTACGTCATA (SEQ ID NO:121),18-25 nucleotides in length and ACTCTTGCCTACGTCATA (SEQ ID NO:122), or19-25 nucleotides in length and CACTCTTGCCTACGTCATA (SEQ ID NO:123).

In some embodiments, the ASO comprises, consists essentially of, orconsists of the sequence CACTCTTGCCTACGTCATAA (SEQ ID NO:115) or asequence at least 90% identical thereto, e.g., at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, theASO comprises, consists essentially of or consists of the sequenceGCACTCTTGCCTACGTCATA (SEQ ID NO:116) or a sequence at least 90%identical thereto, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical.

The ASO may be comprised of deoxyribonucleotides, ribonucleotides, or acombination thereof.

In some embodiments, the ASO comprises at least one non-naturallyoccurring chemical modification. In certain embodiments, at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of thenucleotide linkages are chemically modified. In some embodiments, theASO comprises at least one phosphorothioate linkage. In someembodiments, the ASO comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, or 19 phosphorothioate linkages. In someembodiments, the ASO comprises all phosphorothioate linkages.

In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, or 19 of the nucleotides are chemicallymodified. In some embodiments, the ASO comprises at least one modifiednucleotide at or near the 5′ end and/or the 3′ end, e.g., within 5nucleotides of 5′ end and/or the 3′ end, e.g., at least 1, 2, 3, 4, or 5modified nucleotides. In some embodiments, the ASO comprises at least 3modified nucleotides at each of the 5′ end and the 3′ end, e.g., atleast 4 or at least 5. In some embodiments, at least one of the modifiednucleotides is a 2′-O-methoxyethyl (2′-MOE)-modified nucleotide. In someembodiments, all of the modified nucleotides is a 2′-MOE-modifiednucleotide. In some embodiments, the ASO comprises at least 32′-MOE-modified nucleotides at each of the 5′ end and/or the 3′ end,e.g., at least 4 or at least 5.

In some embodiments, the ASO comprises, consists essentially of, orconsists of a sequence selected from:

a) (SEQ ID NO: 68) C*A*C*T*C*T*T*G*C*C*T*A*C*G*T*C*A*T*A*A; or b)(SEQ ID NO: 69) G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*T*C*A*T*A;wherein * indicates a phosphorothioate linkage and bold indicates a2′-MOE-modified nucleotide. In some embodiments, the ASO comprises,consists essentially of, or consists of a sequence at least 90%identical to one of SEQ ID NO:68 and SEQ ID NO:69, e.g., at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

Another aspect of the invention relates to an ASO targeted to anaturally-occurring human KRAS mRNA encoding a mutation selected fromG12C, G12D, G12V, and G13D, wherein the ASO is 16-25 nucleotides inlength and comprises or consists essentially of a sequence selectedfrom:

-   -   a) TCTTGCCTACGCCACA (SEQ ID NO:117) targeted to a human KRAS        mRNA encoding a G12C mutation;    -   b) TCTTGCCTACGCCATC (SEQ ID NO:118) targeted to a human KRAS        mRNA encoding a G12D mutation;    -   c) TCTTGCCTACGCCAAC (SEQ ID NO:119) targeted to a human KRAS        mRNA encoding a G12V mutation;    -   d) TCTTGCCTACGTCACC (SEQ ID NO:120) targeted to a human KRAS        mRNA encoding a G13D mutation; or    -   e) a sequence at least 90% identical to any one of a) to d);        wherein the antisense oligonucleotide comprises at least one        non-naturally occurring chemical modification.

In some embodiments, the ASO is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to one of SEQ ID NOS:117-120.

In some embodiments, the ASO is 16, 17, 18, 19, 20, 21, 22, 23, 24, or25 nucleotides in length or any range therein. In some embodiments, theASO is 20, 21, or 22 nucleotides in length. In some embodiments, the ASOis 20 nucleotides in length. In certain embodiments, at least 80% of theunspecified nucleotides in the ASO are complementary to a wild-typehuman KRAS gene, e.g., at least 80%, 85%, 90° %, 91%, 92%, 93%, 94%,95%, 96%, 97%. 98%, or 99%. In certain embodiments, at least 80% of theunspecified nucleotides in the ASO are complementary to a mutant humanKRAS gene, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%. In certain embodiments, the ASO is 17-25 nucleotidesin length and comprises an additional nucleotide C at the 5′ end of thesequence of one of SEQ ID NOS:117-120. In certain embodiments, the ASOis 18-25 nucleotides in length and comprises additional nucleotides ACat the 5′ end of the sequence of one of SEQ ID NOS:117-120. In certainembodiments, the ASO is 19-25 nucleotides in length and comprisesadditional nucleotides CAC at the 5′ end of the sequence of one of SEQID NOS:117-120. In certain embodiments, the ASO is 20-25 nucleotides inlength and comprises additional nucleotides GCAC at the 5′ end of thesequence of one of SEQ ID NOS:117-120.

In some embodiments, the antisense oligonucleotide consists of asequence selected from:

-   -   a) GCACTCTTGCCTACGCCACA (SEQ ID NO:124) targeted to a human KRAS        mRNA encoding a G12C mutation;    -   b) GCACTCTTGCCTACGCCATC (SEQ ID NO:125) targeted to a human KRAS        mRNA encoding a G12D mutation;    -   c) GCACTCTTGCCTACGCCAAC (SEQ ID NO:126) targeted to a human KRAS        mRNA encoding a G12V mutation; or    -   d) GCACTCTTGCCTACGTCACC (SEQ ID NO:127) targeted to a human KRAS        mRNA encoding a G13D mutation.

The ASO may be comprised of deoxyribonucleotides, ribonucleotides, or acombination thereof.

In some embodiments, the ASO comprises at least one non-naturallyoccurring chemical modification. In certain embodiments, at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of thenucleotide linkages are chemically modified. In some embodiments, theASO comprises at least one phosphorothioate linkage. In someembodiments, the ASO comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, or 19 phosphorothioate linkages. In someembodiments, the ASO comprises all phosphorothioate linkages.

In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, or 19 of the nucleotides are chemicallymodified. In some embodiments, the ASO comprises at least one modifiednucleotide at or near the 5′ end and/or the 3′ end, e.g., within 5nucleotides of 5′ end and/or the 3′ end, e.g., at least 1, 2, 3, 4, or 5modified nucleotides. In some embodiments, the ASO comprises at leastfive modified nucleotides at each of the 5′ end and the 3′ end. In someembodiments, at least one of the modified nucleotides is a2′-MOE-modified nucleotide. In some embodiments, all of the modifiednucleotides is a 2′-MOE-modified nucleotide. In some embodiments, theASO comprises at least 3 2′-MOE-modified nucleotides at each of the 5′end and/or the 3′ end, e.g., at least 4 or at least 5.

In certain embodiments, the ASO consists of a sequence selected from:

a) (SEQ ID NO: 70) G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*C*A; b)(SEQ ID NO: 71) G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*T*C; c)(SEQ ID NO: 72) G*C*A*C*T*C*T*T*G*C*C*T*A*C'G*C*C*A*A*C; or d)(SEQ ID NO: 73) G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*T*C*A*C*C;wherein * indicates a phosphorothioate linkage and bold indicates a2′-MOE-modified nucleotide. In some embodiments, the ASO comprises,consists essentially of, or consists of a sequence at least 90%identical to one of SEQ ID NOS:70-73, e.g., at least 90%, 91%, 92%, 93%,94%. 95%, 96%, 97%, 98%, or 99% identical.

In some embodiments, at least one modified nucleotide is a lockednucleic acid nucleotide, e.g., at least 1, 2, 3, 4, or 5 modifiednucleotides. The lock nucleic acid may be, without limitation, amethylene bridge connecting the 2′ oxygen and 4′ carbon on the ribose tolock the ribose in the 3′-endo (North) conformation.

In certain embodiments, the ASO consists of a sequence selected from:

a) (SEQ ID NO: 74) G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*C*+A; b)(SEQ ID NO: 75) G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*+T*C; c)(SEQ ID NO: 76) G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*+A*C; or d)(SEQ ID NO: 77) G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*+T*C*A*C*C;wherein * indicates a phosphorothioate linkage, bold indicates a2′-methoxymethyl-modified nucleotide, and + indicates the followingnucleotide is a locked nucleic acid. In some embodiments, the ASOcomprises, consists essentially of, or consists of a sequence at least90% identical to one of SEQ ID NOS:74-76, e.g., at least 90%, 91%. 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.Chemically-Modified siRNAs

One aspect of the invention relates to a siRNA molecule targeted to anaturally-occurring human KRAS mRNA encoding a mutation selected fromG12C, G12D, G12V, and G13D, wherein the siRNA comprises as least onechemical modification. In some embodiments, the siRNA molecule is fullychemically modified. The term “fully chemically-modified” means thatevery nucleotide in the siRNA contains a chemical modification. In someembodiments, each nucleotide in the siRNA molecule is modified with a2′-O-methyl group or a 2′-fluoro group.

In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, or 19 of the nucleotide linkages in the siRNAare chemically modified. In some embodiments, the siRNA comprises atleast one phosphorothioate linkage. In some embodiments, the siRNAcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,or 19 phosphorothioate linkages. In some embodiments, the siRNAcomprises all phosphorothioate linkages.

In certain embodiments, the siRNA molecule comprising at least onechemical modification comprises a sense strand and an antisense strand,wherein the siRNA molecule comprises one of the following pairs ofsequences:

-   -   sense strand of SEQ ID NO:128 and antisense strand of SEQ ID        NO:129;    -   sense strand of SEQ ID NO:130 and antisense strand of SEQ ID        NO:131;    -   sense strand of SEQ ID NO:132 and antisense strand of SEQ ID        NO:133;    -   sense strand of SEQ ID NO:134 and antisense strand of SEQ ID        NO:135;    -   sense strand of SEQ ID NO:136 and antisense strand of SEQ ID        NO:137;    -   sense strand of SEQ ID NO:138 and antisense strand of SEQ ID        NO:139;    -   sense strand of SEQ ID NO:140 and antisense strand of SEQ ID        NO:141;    -   sense strand of SEQ ID NO:142 and antisense strand of SEQ ID        NO:143;    -   sense strand of SEQ ID NO:144 and antisense strand of SEQ ID        NO:145;    -   sense strand of SEQ ID NO:146 and antisense strand of SEQ ID        NO:147;    -   sense strand of SEQ ID NO:148 and antisense strand of SEQ ID        NO:149;    -   sense strand of SEQ ID NO:150 and antisense strand of SEQ ID        NO:151;    -   sense strand of SEQ ID NO:152 and antisense strand of SEQ ID        NO:153;    -   sense strand of SEQ ID NO:154 and antisense strand of SEQ ID        NO:155;    -   sense strand of SEQ ID NO:156 and antisense strand of SEQ ID        NO:157; or    -   sense strand of SEQ ID NO:158 and antisense strand of SEQ ID        NO:159.

In some embodiments, the siRNA comprises, consists essentially of, orconsists of a sequence at least 90% identical to one of SEQ IDNOS:128-159, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical.

TABLE 1 Fully modified siRNA sequences Strand Sequence (5′-3′) SEQ ID NOS GAGCUUGUGGCGUAGGCAAGA 128 AS UUGCCUACGCCACAAGCUCCA 129 SGAGCUGAUGGCGUAGGCAAGA 130 AS UUGCCUACGCCAUCAGCUCCA 131 SGAGCUGGUGACGUAGGCAAGA 132 AS UUGCCUACGUCACCAGCUCCA 133 SAGUUGGAGCUUGUGGCGUAGG 134 AS UACGCCACAAGCUCCAACUAC 135 SAGUUGGAGCUGAUGGCGUAGG 136 AS UACGCCAUCAGCUCCAACUAC 137 SAGUUGGAGCUGGUGACGUAGG 138 AS UACGUCACCAGCUCCAACUAC 139 SGGUAGUUGGAGCUGGUGACGU 140 AS GUCACCAGCUCCAACUACCAC 141 SAGUUGGAGCUGUUGGCGUAGG 142 AS UACGCCAACAGCUCCAACUAC 143 SGAGCUGUUGGCGUAGGCAAGA 144 AS UUGCCUACGCCAACAGCUCCA 145 SGAGCUGAUGGCGUAGGCAAGA 146 AS UUGCCUACGCCAUCAGCUCCA 147 SGAGCUGGUGACGUAGGCAAGA 148 AS UUGCCUACGUCACCAGCUCCA 149 SAGUUGGAGCUUGUGGCGUAGG 150 AS UACGCCACAAGCUCCAACUAC 151 SAGUUGGAGCUGAUGGCGUAGG 152 AS UACGCCAUCAGCUCCAACUAC 153 SAGUUGGAGCUGGUGACGUAGG 154 AS UACGUCACCAGCUCCAACUAC 155 SAGUUGGAGCUGUUGGCGUAGG 156 AS UACGCCAACAGCUCCAACUAC 157 SGAGCUGUUGGCGUAGGCAAGA 158 AS UUGCCUACGCCAACAGCACCA 159 S-sense strandAS-antisense strand

In certain embodiments, the siRNA molecule is fully chemically modifiedand comprises a sense strand and an antisense strand, wherein the siRNAmolecule comprises one of the following pairs of sequences:

-   -   sense strand of SEQ ID NO:78 and antisense strand of SEQ IDI        NO:79;    -   sense strand of SEQ ID NO:80 and antisense strand of SEQ ID        NO:81;    -   sense strand of SEQ ID NO:82 and antisense strand of SEQ ID        NO:83;    -   sense strand of SEQ ID NO:84 and antisense strand of SEQ ID        NO:85;    -   sense strand of SEQ ID NO:86 and antisense strand of SEQ ID        NO:87;    -   sense strand of SEQ ID NO:88 and antisense strand of SEQ ID        NO:89;    -   sense strand of SEQ ID NO:90 and antisense strand of SEQ ID        NO:91;    -   sense strand of SEQ ID NO:92 and antisense strand of SEQ IDI        NO:93;    -   sense strand of SEQ ID NO:94 and antisense strand of SEQ ID        NO:95;    -   sense strand of SEQ ID NO:96 and antisense strand of SEQ ID        NO:97;    -   sense strand of SEQ ID NO:98 and antisense strand of SEQ ID        NO:99;    -   sense strand of SEQ ID NO:100 and antisense strand of SEQ ID        NO:101;    -   sense strand of SEQ ID NO:102 and antisense strand of SEQ ID        NO:103;    -   sense strand of SEQ ID NO:104 and antisense strand of SEQ ID        NO:105;    -   sense strand of SEQ ID) NO:106 and antisense strand of SEQ ID        NO:107; or    -   sense strand of SEQ ID) NO:108 and antisense strand of SEQ ID        NO:109.

In some embodiments, the siRNA comprises, consists essentially of, orconsists of a sequence at least 90% identical to one of SEQ IDNOS:78-109, e.g., at least 90%/e, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical.

Another aspect of the invention relates to an siRNA molecule targeted tohuman KRAS mRNA, wherein the sense strand of the siRNA comprises,consists essentially of, or consists of the sequence of SEQ ID NO:50 orSEQ ID NO:51, and wherein the siRNA comprises at least one non-naturallyoccurring chemical modification. In some embodiments, at least 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 of the nucleotides in the siRNA are chemicallymodified. In some embodiments, the siRNA is fully chemically-modified.In some embodiments, each nucleotide in the siRNA molecule is modifiedwith a 2′-O-methyl group or a 2′-fluoro group.

In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, or 19 of the nucleotide linkages in the siRNAare chemically modified. In some embodiments, the siRNA comprises atleast one phosphorothioate linkage. In some embodiments, the siRNAcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,or 19 phosphorothioate linkages. In some embodiments, the siRNAcomprises all phosphorothioate linkages.

In certain embodiments, the fully chemically-modified siRNA moleculecomprises a sense strand and an antisense strand, wherein the siRNAmolecule comprises one of the following pairs of sequences:

-   -   sense strand of SEQ ID NO:110 and antisense strand of SEQ ID        NO:111; or    -   sense strand of SEQ ID NO:112 and antisense strand of SEQ ID        NO:113.

In some embodiments, the siRNA comprises, consists essentially of, orconsists of a sequence at least 90% identical to one of SEQ IDNOS:110-113, e.g., at least 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%,or 99% identical.

The double stranded RNA molecule, ASO, or chemically-modified siRNAmolecule may be constructed using chemical synthesis and enzymaticligation reactions by procedures known in the art. For example, a doublestranded RNA, ASO, or chemically-modified siRNA molecule may bechemically synthesized using naturally occurring nucleotides or variousmodified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability of the duplex formedbetween the double stranded RNA, ASO, or chemically-modified siRNAmolecule and target nucleotide sequences, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used. Examplesof modified nucleotides which can be used to generate the doublestranded RNA, ASO, or chemically-modified siRNA molecule include, butare not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomet-hyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine. 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the double stranded RNA or ASO canbe produced using an expression vector into which a nucleic acidencoding the double stranded RNA or ASO has been cloned.

The double stranded RNA, ASO, or chemically-modified siRNA molecule canfurther include nucleotide sequences wherein at least one, or all, ofthe internucleotide bridging phosphate residues are modified phosphates,such as methyl phosphonates, methyl phosphonothioates,phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. Forexample, every one or every other one of the internucleotide bridgingphosphate residues can be modified as described. In another non-limitingexample, the double stranded RNA, ASO, or chemically-modified siRNAmolecule is a nucleotide sequence in which at least one, or all, of thenucleotides contain a 2′ lower alkyl moiety (e.g., C₁-C₄, linear orbranched, saturated or unsaturated alkyl, such as methyl, ethyl,ethenyl, propyl. 1-propenyl, 2-propenyl, and isopropyl). In anotherexample, one or more of the nucleotides may be a 2′-fluoro nucleotide, a2-O-methyl nucleotide, or a locked nucleic acid nucleotide. For example,every one or every other one of the nucleotides can be modified asdescribed. See also, Furdon et al., Nucleic Acids Res. 17:9193 (1989);Agrawal el al., Proc. Natl. Acad. Sci. USA 87:1401 (1990); Baker et al.,Nucleic Acids Res. 18:3537 (1990); Sproat et al., Nucleic Acids Res.17:3373 (1989); Walder and Walder, Proc. Natl. Acad. Sd. USA 85:5011(1988); incorporated by reference herein in their entireties for theirteaching of methods of making polynucleotide molecules, including thosecontaining modified nucleotide bases).

The invention further relates to a nucleic acid construct comprising theRNA molecule or ASO of the invention. The invention further relates to anucleic acid construct encoding the RNA molecule or ASO of the inventionand a nucleic acid construct comprising the nucleic acid moleculeencoding the RNA molecule or ASO. In each of these embodiments, thenucleic acid construct may be a vector or a plasmid, e.g., an expressionvector.

Another aspect of the invention relates to a composition comprising theRNA molecule, ASO, chemically-modified siRNA molecule, or nucleic acidconstruct of the invention and another component, e.g., a suitablecarrier. In some embodiments, the composition comprises two or more ofthe RNA molecules, ASOs, chemically-modified siRNA molecules, or nucleicacid constructs of the invention, wherein the two or more RNA molecules,ASO, or chemically-modified siRNA molecule each comprise a differentantisense strand. In certain embodiments, the two or more RNA moleculesare present on the same nucleic acid construct, on different nucleicacid constructs or any combination thereof.

In some embodiments, the composition is a pharmaceutical compositioncomprising the RNA molecule(s), ASO(s), chemically-modified siRNAmolecule(s), or nucleic acid construct(s) of the invention and apharmaceutically acceptable carrier.

It is understood that the compositions of this invention can comprise,consist essentially of, or consist of any of the RNA molecules, ASOs,chemically-modified siRNA molecules, and nucleic acid constructs in anycombination and in any ratio relative to one another. Furthermore, by“two or more” is meant 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., up to a totalnumber of RNA molecules, ASOs, chemically-modified siRNA molecules, andnucleic acid constructs of this invention. In some embodiments, thecompositions comprise, consist essentially of or consist of the RNAmolecules of SEQ ID NO:1 and SEQ ID NO:3.

In some aspects of the invention, the composition or pharmaceuticalcomposition further comprises additional components that enhance thedelivery of the RNA molecule(s), ASO(s), chemically-modified siRNAmolecule(s), or nucleic acid construct(s) of the invention to a subject,e.g., by enhancing the stability of the RNA molecule(s), ASO(s),chemically-modified siRNA molecule(s), or nucleic acid construct(s). Insome embodiments, the additional component may be a particle, e.g., amicroparticle or nanoparticle. In some embodiments, the particle is alipid particle, e.g., a liposome, e.g., a microliposome or ananoliposome. The liposome, microliposome, or nanoliposome may containany components known in the art to be suitable for preparing liposomes.In some embodiments, the liposome comprises1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Liposomes may beprepared by methods known in the art, e.g., as described in Pecot etal., Mol. Cancer Ther. 13:2876 (2014), incorporated by reference hereinin its entirety. In some embodiments, the RNA molecule is formed into astable nucleic acid lipid particle (SNALP), e.g., using particles suchas those provided by Arbutus Biopharma (Doylestown, Pa.). In certainembodiments, the lipid particle comprises, consists essentially of, orconsists of cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC). PEG-cDMA or PEG-cDSA, and1,2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane (DLinDMA) (see Judge etal., J. Clin. Invest. 119:661 (2009)). In some embodiments, the lipidparticle comprises two or more of the RNA molecules, ASOs, orchemically-modified siRNA molecules of the invention. e.g., the RNAmolecules of SEQ ID NO:1 and SEQ ID NO:3. In some embodiments, theadditional component is a targeted delivery moiety to which the RNAmolecule(s), ASO(s), chemically-modified siRNA molecule(s), or nucleicacid construct(s) or covalently or noncovalently conjugated, e.g.,ligands, aptamers, or monoclonal antibodies.

The present invention encompasses cells comprising the RNA moleculesand/or nucleic acid constructs of the invention. Thus, in someembodiments, the present invention provides a transformed cellcomprising a RNA molecule and/or a nucleic acid construct and/or acomposition of this invention, wherein the transformed cell has reducedexpression of mutant KRAS as compared to a control cell.

Methods

Various methods are provided herein, employing the nucleic acidmolecules, nucleic acid constructs, and/or compositions of thisinvention. Thus, in one aspect, the present invention provides a methodof inhibiting expression of a mutant human KRAS gene comprising one ormore of the missense mutations G12C, G12D, G12V, and G13D in a cell, themethod comprising contacting the cell with the RNA molecule, ASO,chemically-modified siRNA molecule, nucleic acid construct, composition,and/or pharmaceutical composition of the invention, thereby inhibitingexpression of the mutant human KRAS gene in the cell.

Also provided herein is a method of treating cancer in a subject in needthereof, wherein the cancer comprises a mutant human KRAS genecomprising one or more of the missense mutations G12C, G12D, G12V, andG13D, the method comprising delivering to the subject the RNA molecule,ASO, chemically-modified siRNA molecule, nucleic acid construct,composition, and/or pharmaceutical composition of the invention, therebytreating cancer in the subject. A cancer comprising a mutant human KRASgene comprising one or more of the missense mutations G12C, G12D, G12V,and G13D is a cancer, e.g., a tumor in which one or more cells expressthe mutant KRAS gene.

In one embodiment of each of these aspects, the subject may be one thathas been diagnosed with cancer. In another embodiment, the subject maybe one that is at risk of developing cancer (e.g., predisposed due tohereditary factors, smoking, viral infection, exposure to chemicals,etc.). In a further embodiment, the subject may be one that has beenidentified as carrying a mutant KRAS gene and has or has not beendiagnosed with cancer.

The double stranded RNA, ASO, or chemically-modified siRNA molecule ofthe invention can be delivered directly into a cell by any method knownin the art, e.g., by transfection or microinjection, e.g., as part of acomposition comprising lipid particles. In other embodiments, the doublestranded RNA or ASO can be delivered to a subject in the form ofpolynucleotides encoding the RNA or ASO to produce expression of thedouble stranded RNA or ASO within the cells of the subject. Thoseskilled in the art will appreciate that the isolated polynucleotidesencoding the RNAs or ASOs of the invention will typically be associatedwith appropriate expression control sequences, e.g.,transcription/translation control signals and polyadenylation signals.

It will further be appreciated that a variety of promoter/enhancerelements can be used depending on the level and tissue-specificexpression desired. The promoter can be constitutive or inducible,depending on the pattern of expression desired. The promoter can benative or foreign and can be a natural or a synthetic sequence. Byforeign, it is intended that the transcriptional initiation region isnot found in the wild-type host into which the transcriptionalinitiation region is introduced. The promoter is chosen so that it willfunction in the target cell(s) of interest.

To illustrate, the RNA or ASO coding sequence can be operativelyassociated with a cytomegalovirus (CMV) major immediate-early promoter,an albumin promoter, an Elongation Factor 1-α (EF1-α) promoter, a PγKpromoter, a MFG promoter, or a Rous sarcoma virus promoter.

Inducible promoter/enhancer elements include hormone-inducible andmetal-inducible elements, and other promoters regulated by exogenouslysupplied compounds, including without limitation, the zinc-induciblemetallothionein (MT) promoter; the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter; the T7 polymerase promoter system(see WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl.Acad. Sci. USA 93:3346 (1996)); the tetracycline-repressible system(Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547 (1992)); thetetracycline-inducible system (Gossen et al., Science 268:1766 (1995);see also Harvey et al., Curr. Opin. Chem. Biol. 2:512 (1998)); theRU486-inducible system (Wang et al., Nat. Biotech. 15:239 (1997); Wanget al., Gene Ther., 4:432(1997)); and the rapamycin-inducible system(Magari et al., J. Clin. Invest. 100:2865 (1997)).

Other tissue-specific promoters or regulatory promoters include, but arenot limited to, promoters that typically confer tissue-specificity inneurons. These include, but are not limited to, promoters for synapsin1, tubulin α1, platelet-derived growth factor B-chain, tyrosinehydroxylase, neuron-specific enolase, and neurofilaments. Skeletalmuscle cell promoters include, but are not limited to, promoters forβ-actin, Pitx3, creatine kinase, and myosin light chain. Cardiac musclecell promoters include, but are not limited to, promoters for cardiacactin, cardiac troponin T, troponin C, myosin light chain-2, andα-myosin heavy chain. Islet (beta) cell promoters include, but are notlimited to, glucokinase, gastrin, insulin, and islet amyloidpolypeptide.

Moreover, specific initiation signals are generally required forefficient translation of inserted RNA or ASO coding sequences. Thesetranslational control sequences, which can include the ATG initiationcodon and adjacent sequences, can be of a variety of origins, bothnatural and synthetic.

The isolated nucleic acid encoding the double stranded RNA or ASO can beincorporated into an expression vector. Expression vectors compatiblewith various host cells are well known in the art and contain suitableelements for transcription and translation of nucleic acids. Typically,an expression vector contains an “expression cassette,” which includes,in the 5′ to 3′ direction, a promoter, a coding sequence encoding adouble stranded RNA operatively associated with the promoter, and,optionally, a termination sequence including a stop signal for RNApolymerase and a polyadenylation signal for polyadenylase.

Non-limiting examples of animal and mammalian promoters known in the artinclude, but are not limited to, the SV40 early (SV40e) promoter region,the promoter contained in the 3′ long terminal repeat (LTR) of Roussarcoma virus (RSV), the promoters of the ElA or major late promoter(MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) earlypromoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter,baculovirus IE1 promoter, elongation factor 1 alpha (EF1) promoter,phosphoglycerate kinase (PGK) promoter, ubiquitin (Ubc) promoter, analbumin promoter, the regulatory sequences of the mousemetallothionein-L promoter and transcriptional control regions, theubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like),the promoters of the intermediate filaments (desmin, neurofilaments,keratin, GFAP, and the like), the promoters of therapeutic genes (of theMDR, CFTR or factor VIII type, and the like), and pathogenesis and/ordisease-related promoters. In addition, any of these expressionsequences of this invention can be modified by addition of enhancerand/or regulatory sequences and the like.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor I (EF1) enhancer, yeast enhancers, viral geneenhancers, and the like.

Termination control regions, i.e., terminator or polyadenylationsequences, may be derived from various genes native to the preferredhosts. In some embodiments of the invention, the termination controlregion may comprise or be derived from a synthetic sequence, a syntheticpolyadenylation signal, an SV40 late polyadenylation signal, an SV40polyadenylation signal, a bovine growth hormone (BGH) polyadenylationsignal, viral terminator sequences, or the like.

It will be apparent to those skilled in the art that any suitable vectorcan be used to deliver the polynucleotide to a cell or subject. Thevector can be delivered to cells in vivo. In other embodiments, thevector can be delivered to cells ex vivo, and then cells containing thevector are delivered to the subject. The choice of delivery vector canbe made based on a number of factors known in the art, including age andspecies of the target host, in vitro versus in vivo delivery, level andpersistence of expression desired, intended purpose (e.g., for therapyor screening), the target cell or organ, route of delivery, size of theisolated polynucleotide, safety concerns, and the like.

Suitable vectors include, but are not limited to, plasmid vectors, viralvectors (e.g., retrovirus, alphavirus; vaccinia virus; adenovirus,adeno-associated virus and other parvoviruses, lentivirus, poxvirus, orherpes simplex virus), lipid vectors, poly-lysine vectors, syntheticpolyamino polymer vectors, and the like.

Any viral vector that is known in the art can be used in the presentinvention. Protocols for producing recombinant viral vectors and forusing viral vectors for nucleic acid delivery can be found in Ausubel etal., Current Protocols in Molecular Biology (Green PublishingAssociates, Inc. and John Wiley & Sons. Inc., New York) and otherstandard laboratory manuals (e.g., Vectors for Gene Therapy. In: CurrentProtocols in Human Genetics. John Wiley and Sons, Inc.: 1997).

Non-viral transfer methods can also be employed. Many non-viral methodsof nucleic acid transfer rely on normal mechanisms used by mammaliancells for the uptake and intracellular transport of macromolecules. Inparticular embodiments, non-viral nucleic acid delivery systems rely onendocytic pathways for the uptake of the nucleic acid molecule by thetargeted cell. Exemplary nucleic acid delivery systems of this typeinclude liposomal derived systems, poly-lysine conjugates, andartificial viral envelopes.

In particular embodiments, plasmid vectors are used in the practice ofthe present invention. For example, naked plasmids can be introducedinto muscle cells by injection into the tissue. Expression can extendover many months, although the number of positive cells is typically low(Wolff et al., Science 247:247 (1989)). Cationic lipids have beendemonstrated to aid in introduction of nucleic acids into some cells inculture (Felgner and Ringold, Nature 337:387 (1989)). Injection ofcationic lipid plasmid DNA complexes into the circulation of mice hasbeen shown to result in expression of the DNA in lung (Brigham et al.,Am. J. Med. Sci. 298:278 (1989)). One advantage of plasmid DNA is thatit can be introduced into non-replicating cells.

In a representative embodiment, a nucleic acid molecule (e.g., aplasmid) can be entrapped in a lipid particle bearing positive chargeson its surface and, optionally, tagged with antibodies against cellsurface antigens of the target tissue (Mizuno et al., No Shinkei Geka20:547 (1992); PCT publication WO 91/06309; Japanese patent application1047381; and European patent publication EP-A-43075).

Liposomes that consist of amphiphilic cationic molecules are useful asnon-viral vectors for nucleic acid delivery in vitro and in vivo(reviewed in Crystal, Science 270:404 (1995); Blaese et al., Cancer GeneTher. 2:291 (1995); Behr et al., Bioconjugate Chem. 5:382 (1994); Remyet al., Bioconjugate Chem. 5:647 (1994); and Gao et al., Gene Therapy2:710 (1995)). The positively charged liposomes are believed to complexwith negatively charged nucleic acids via electrostatic interactions toform lipid:nucleic acid complexes. The lipid:nucleic acid complexes haveseveral advantages as nucleic acid transfer vectors. Unlike viralvectors, the lipid:nucleic acid complexes can be used to transferexpression cassettes of essentially unlimited size. Since the complexeslack proteins, they can evoke fewer immunogenic and inflammatoryresponses. Moreover, they cannot replicate or recombine to form aninfectious agent and have low integration frequency. A number ofpublications have demonstrated that amphiphilic cationic lipids canmediate nucleic acid delivery in vivo and in vitro (Feigner et al.,Proc. Natl. Acad. Sci. USA 84:7413 (1987); Loeffler et al., Meth.Enzymol. 217:599 (1993); Feigner et al., J. Biol. Chem. 269-2550(1994)).

Several groups have reported the use of amphiphilic cationiclipid-nucleic acid complexes for in vivo transfection both in animalsand in humans (reviewed in Gao et al., Gene Therapy 2:710 (1995); Zhu etal., Science 261:209 (1993); and Thierry et al., Proc. Natl. Acad. Sci.USA 92:9742 (1995)). U.S. Pat. No. 6,410,049 describes a method ofpreparing cationic lipid-nucleic acid complexes that have a prolongedshelf life.

Nuclear localization signals can also be used to enhance the targetingof the double stranded RNA or expression vector into the proximity ofthe nucleus and/or its entry into the nucleus. Such nuclear localizationsignals can be a protein or a peptide such as the SV40 large Tag NLS orthe nucleoplasmin NLS. These nuclear localization signals interact witha variety of nuclear transport factors such as the NLS receptor(karyopherin alpha) which then interacts with karyopherin beta.

Expression vectors can be designed for expression of stranded RNA orASOs in prokaryotic or eukaryotic cells. For example, stranded RNA orASOs can be expressed in bacterial cells such as E. coli, insect cells(e.g., the baculovirus expression system), yeast cells, plant cells ormammalian cells. Some suitable host cells are discussed further inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Examples of bacterial vectors include,but are not limited to, pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10,phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A,pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5(Pharmacia). Examples of vectors for expression in the yeast S.cerevisiae include pYepSecl (Baldari et al., EMBO J. 6:229 (1987)), pMFa(Kurjan and Herskowitz, Cell 30:933 (1982)), pJRY88 (Schultz et al.,Gene 54:113 (1987)), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Non-limiting examples of baculovirus vectors available forexpression of nucleic acids to produce proteins in cultured insect cells(e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell.Biol. 3:2156(1983)) and the pVL series (Lucklow and Summers Virology170:31 (1989)).

Examples of mammalian expression vectors include pWLNEO, pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, PBPV, pMSG, PSVL (Pharmacia), pCDM8 (Seed,Nature 329:840 (1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187(1987)). When used in mammalian cells, the expression vector's controlfunctions are often provided by viral regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus and Simian Virus 40.

Viral vectors have been used in a wide variety of gene deliveryapplications in cells, as well as living animal subjects. Viral vectorsthat can be used include, but are not limited to, retrovirus,lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus,vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus,geminivirus, and caulimoviruses vectors. Non-limiting examples ofnon-viral vectors include plasmids, liposomes, electrically chargedlipids (cytofectins), nucleic acid-protein complexes, and biopolymers.In addition to a nucleic acid of interest, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(delivery to specific tissues, duration of expression, etc.).

In addition to the regulatory control sequences discussed above, therecombinant expression vector can contain additional nucleotidesequences. For example, the recombinant expression vector can encode aselectable marker gene to identify host cells that have incorporated thevector.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acids (e.g.,DNA and RNA) into a host cell, including, but are not limited to,calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, electroporation,microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cellsonication, gene bombardment using high velocity microprojectiles, andviral-mediated transfection. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al., MolecularCloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

If stable integration is desired, often only a small fraction of cells(in particular, mammalian cells) integrate the foreign DNA into theirgenome. In order to identify and select integrants, a nucleic acid thatencodes a selectable marker (e.g., resistance to antibiotics) can beintroduced into the host cells along with the nucleic acid of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acids encodinga selectable marker can be introduced into a host cell on the samevector as that comprising the nucleic acid of interest or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

In one embodiment, the double stranded RNA, ASO, or chemically-modifiedsiRNA molecule of the invention is administered directly to the subject.Generally, the compounds of the invention will be suspended in apharmaceutically-acceptable carrier (e.g., physiological saline) andadministered orally, topically, or by intravenous infusion, or injectedsubcutaneously, intramuscularly, intracranially, intrathecally,intraperitoneally, intrarectally, intravaginally, intranasally,intragastrically, intratracheally, or intrapulmonarily. They arepreferably delivered directly to the site of the disease or disorder,such as the lung, intestine, or pancreas. The dosage required depends onthe choice of the route of administration; the nature of theformulation; the nature of the patient's illness; the subject's size,weight, surface area, age, and sex; other drugs being administered; andthe judgment of the attending physician. Suitable dosages are in therange of 0.01-100.0 μg/kg. Wide variations in the needed dosage are tobe expected in view of the differing efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by i.v. injection (e.g., 2-,3-, 4-, 6-. 8-, 10-; 20-, 50-, 100-, 150-, or more fold). Variations inthese dosage levels can be adjusted using standard empirical routinesfor optimization as is well understood in the art. Administrations canbe single or multiple. Encapsulation of the inhibitor in a suitabledelivery vehicle (e.g., polymeric microparticles or implantable devices)may increase the efficiency of delivery, particularly for oral delivery.

According to certain embodiments, the double stranded RNA, ASO, orchemically-modified siRNA molecule can be targeted to specific cells ortissues in vivo. Targeting delivery vehicles, including liposomes andviral vector systems are known in the at. For example, a liposome can bedirected to a particular target cell or tissue by using a targetingagent, such as an antibody, soluble receptor or ligand, incorporatedwith the liposome, to target a particular cell or tissue to which thetargeting molecule can bind. Targeting liposomes are described, forexample, in Ho et al., Biochemistry 25:5500 (1986); Ho et al., J. Biol.Chem. 262:13979 (1987); Ho et al., J. Biol. Chem. 262:13973 (1987); andU.S. Pat. No. 4,957,735 to Huang et al., each of which is incorporatedherein by reference in its entirety). Enveloped viral vectors can bemodified to deliver a nucleic acid molecule to a target cell bymodifying or substituting an envelope protein such that the virusinfects a specific cell type. In adenoviral vectors, the gene encodingthe attachment fibers can be modified to encode a protein domain thatbinds to a cell-specific receptor. Herpesvirus vectors naturally targetthe cells of the central and peripheral nervous system. Alternatively,the route of administration can be used to target a specific cell ortissue. For example, intracoronary administration of an adenoviralvector has been shown to be effective for the delivery of a gene tocardiac myocytes (Maurice et al., J. Clin. Invest. 104:21 (1999)).Intravenous delivery of cholesterol-containing cationic liposomes hasbeen shown to preferentially target pulmonary tissues (Liu et al.,Nature Biotechnol. 15:167 (1997)), and effectively mediate transfer andexpression of genes in vivo. Other examples of successful targeted invivo delivery of nucleic acid molecules are known in the art. Finally, arecombinant nucleic acid molecule can be selectively (i.e.,preferentially, substantially exclusively) expressed in a target cell byselecting a transcription control sequence, and preferably, a promoter,which is selectively induced in the target cell and remainssubstantially inactive in non-target cells.

The double stranded RNA, ASO, or chemically-modified siRNA molecule ofthe present invention can optionally be delivered in conjunction withother therapeutic agents. The additional therapeutic agents can bedelivered concurrently with the double stranded RNA, ASO, orchemically-modified siRNA molecule of the invention. As used herein, theword “concurrently” means sufficiently close in time to produce acombined effect (that is, concurrently can be simultaneously, or it canbe two or more events occurring within a short time period before orafter each other). In one embodiment, the double stranded RNA, ASO, orchemically-modified siRNA molecule of the invention are administered inconjunction with agents useful for treating cancer, such as: 1) vincaalkaloids (e.g., vinblastine, vincristine); 2) epipodophyllotoxins(e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin(actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin,bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4)enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g.,interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatinand carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8)substituted ureas (e.g., hydroxyurea); 9) methylhydrazine derivatives(e.g., procarbazine (N-methylhydrazine; MIH)); 10) adrenocorticalsuppressants (e.g., mitotane (o,p′-DDD) and aminoglutethimide); 11)adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g.,hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrolacetate); 13) estrogens (e.g., diethylstilbestrol and ethinylestradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g.,testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g.,flutamide); and 17) gonadotropin-releasing hormone analogs (e.g.,leuprolide). In another embodiment, the compounds of the invention areadministered in conjunction with anti-angiogenesis agents, such asantibodies to VEGF (e.g., bevacizumab (AVASTIN), ranibizumab (LUCENTIS))and other promoters of angiogenesis (e.g., bFGF, angiopoietin-1),antibodies to alpha-v/beta-3 vascular integrin (e.g., VITAXIN),angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNF alphaconjugate, cyclophosphamide, combretastatin A4 phosphate,dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurin,paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation(Abraxane), soy isoflavone (Genistein), tamoxifen citrate, thalidomide,ADH-1 (EXHERIN), AG-013736, AMG-706, AZD2171, sorafenib tosylate,BMS-582664, CHIR-265, pazopanib, PI-88, vatalanib, everolimus, suramin,sunitinib malate, XL184, ZD6474, ATN-161, cilenigtide, and celecoxib, orany combination thereof.

The term “cancer,” as used herein, refers to any benign or malignantabnormal growth of cells. Examples include, without limitation, breastcancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, coloncancer, melanoma, malignant melanoma, ovarian cancer, brain cancer,primary brain carcinoma, head-neck cancer, glioma, glioblastoma, livercancer, bladder cancer, non-small cell lung cancer, head or neckcarcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma,small-cell lung carcinoma, Wilmis' tumor, cervical carcinoma, testicularcarcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma,colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroidcarcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenalcarcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortexcarcinoma, malignant pancreatic insulinoma, malignant carcinoidcarcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia,cervical hyperplasia, leukemia, acute lymphocytic leukemia, chroniclymphocytic leukemia, acute myelogenous leukemia, chronic myelogenousleukemia, chronic granulocytic leukemia, acute granulocytic leukemia,hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma,polycythemia vera, essential thrombocytosis, Hodgkin's disease,non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primarymacroglobulinemia, and retinoblastoma. In some embodiments, the canceris selected from the group of tumor-forming cancers.

Pharmaceutical Compositions

As a further aspect, the invention provides pharmaceutical formulationsand methods of administering the same to achieve any of the therapeuticeffects (e.g., treatment of cancer) discussed above. The pharmaceuticalformulation may comprise any of the reagents discussed above in apharmaceutically acceptable carrier.

By “pharmaceutically acceptable” it is meant a material that is notbiologically or otherwise undesirable, i.e., the material can beadministered to a subject without causing any undesirable biologicaleffects such as toxicity.

The formulations of the invention can optionally comprise medicinalagents, pharmaceutical agents, carriers, adjuvants, dispersing agents,diluents, and the like.

The double stranded RNA, ASO, chemically-modified siRNA molecule, ornucleic acid construct of the invention can be formulated foradministration in a pharmaceutical carrier in accordance with knowntechniques. See, e.g., Remington, The Science And Practice of Pharmacy(9^(th) Ed. 1995). In the manufacture of a pharmaceutical formulationaccording to the invention, the double stranded RNA, ASO, orchemically-modified siRNA molecule (including the physiologicallyacceptable salts thereof) is typically admixed with, inter alia, anacceptable carrier. The carrier can be a solid or a liquid, or both, andis preferably formulated with the double stranded RNA, ASO, orchemically-modified siRNA molecule as a unit-dose formulation, forexample, a tablet, which can contain from 0.01 or 0.5% to 95% or 99% byweight of the double stranded RNA, ASO, or chemically-modified siRNAmolecule. One or more double stranded RNAs, ASOs, or chemically-modifiedsiRNA molecules can be incorporated in the formulations of theinvention, which can be prepared by any of the well-known techniques ofpharmacy.

A further aspect of the invention is a method of treating subjects invivo, comprising administering to a subject a pharmaceutical compositioncomprising a double stranded RNA, ASO, or chemically-modified siRNAmolecule of the invention in a pharmaceutically acceptable carrier,wherein the pharmaceutical composition is administered in atherapeutically effective amount. Administration of the double strandedRNA, ASO, or chemically-modified siRNA molecule of the present inventionto a human subject or an animal in need thereof can be by any meansknown in the art for administering compounds.

Non-limiting examples of formulations of the invention include thosesuitable for oral, rectal, buccal (e.g., sub-lingual), vaginal,parenteral (e.g., subcutaneous, intramuscular including skeletal muscle,cardiac muscle, diaphragm muscle and smooth muscle, intradermal,intravenous, intraperitoneal), topical (i.e., both skin and mucosalsurfaces, including airway surfaces), intranasal, transdermal,intraarticular, intracranial, intrathecal, and inhalationadministration, administration to the liver by intraportal delivery, aswell as direct organ injection (e.g., into the liver, into a limb, intothe brain or spinal cord for delivery to the central nervous system,into the pancreas, or into a tumor or the tissue surrounding a tumor).

The most suitable route in any given case will depend on the nature andseverity of the condition being treated and on the nature of theparticular compound which is being used. In some embodiments, it may bedesirable to deliver the formulation locally to avoid any side effectsassociated with systemic administration. For example, localadministration can be accomplished by direct injection at the desiredtreatment site, by introduction intravenously at a site near a desiredtreatment site (e.g., into a vessel that feeds a treatment site). Insome embodiments, the formulation can be delivered locally to ischemictissue. In certain embodiments, the formulation can be a slow releaseformulation, e.g., in the form of a slow release depot.

For injection, the carrier will typically be a liquid, such as sterilepyrogen-free water, pyrogen-free phosphate-buffered saline solution,bacteriostatic water, or Cremophor EL[R](BASF, Parsippany, N.J.). Forother methods of administration, the carrier can be either solid orliquid.

For oral administration, the compound can be administered in soliddosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Compounds can beencapsulated in gelatin capsules together with inactive ingredients andpowdered carriers, such as glucose, lactose, sucrose, mannitol, starch,cellulose or cellulose derivatives, magnesium stearate, stearic acid,sodium saccharin, talcum, magnesium carbonate and the like. Examples ofadditional inactive ingredients that can be added to provide desirablecolor, taste, stability, buffering capacity, dispersion or other knowndesirable features are red iron oxide, silica gel, sodium laurylsulfate, titanium dioxide, edible white ink and the like. Similardiluents can be used to make compressed tablets. Both tablets andcapsules can be manufactured as sustained release products to providefor continuous release of medication over a period of hours. Compressedtablets can be sugar coated or film coated to mask any unpleasant tasteand protect the tablet from the atmosphere, or enteric-coated forselective disintegration in the gastrointestinal tract. Liquid dosageforms for oral administration can contain coloring and flavoring toincrease patient acceptance.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the compound in a flavored base, usually sucrose andacacia or tragacanth; and pastilles comprising the compound in an inertbase such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the compound, which preparations are preferably isotonicwith the blood of the intended recipient. These preparations can containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient. Aqueousand non-aqueous sterile suspensions can include suspending agents andthickening agents. The formulations can be presented in unit/dose ormulti-dose containers, for example sealed ampoules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, saline orwater-for-injection immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a compound of theinvention, in a unit dosage form in a sealed container. The compound orsalt is provided in the form of a lyophilizate which is capable of beingreconstituted with a suitable pharmaceutically acceptable carrier toform a liquid composition suitable for injection thereof into a subject.The unit dosage form typically comprises from about 10 ng to about 10grams of the compound or salt. When the compound or salt issubstantially water-insoluble, a sufficient amount of emulsifying agentwhich is pharmaceutically acceptable can be employed in sufficientquantity to emulsify the compound or salt in an aqueous carrier. Onesuch useful emulsifying agent is phosphatidyl choline.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These can be prepared by admixing thecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which can be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration can be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration can also be delivered byiontophoresis (see, for example, Tyle, Pharm. Res. 3:318 (1986)) andtypically take the form of an optionally buffered aqueous solution ofthe compound. Suitable formulations comprise citrate or bis\tris buffer(pH 6) or ethanol/water and contain from 0.1 to 0.2M of the compound.

The compound can alternatively be formulated for nasal administration orotherwise administered to the lungs of a subject by any suitable means,e.g., administered by an aerosol suspension of respirable particlescomprising the compound, which the subject inhales. The respirableparticles can be liquid or solid. The term “aerosol” includes anygas-borne suspended phase, which is capable of being inhaled into thebronchioles or nasal passages. Specifically, aerosol includes agas-borne suspension of droplets, as can be produced in a metered doseinhaler or nebulizer, or in a mist sprayer. Aerosol also includes a drypowder composition suspended in air or other carrier gas, which can bedelivered by insufflation from an inhaler device, for example. SeeGanderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood(1987); Gonda (1990) Critical Reviews in Therapeutic Drug CarrierSystems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth.27:143 (1992).

Aerosols of liquid particles comprising the compound can be produced byany suitable means, such as with a pressure-driven aerosol nebulizer oran ultrasonic nebulizer, as is known to those of skill in the art. See,e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles comprisingthe compound can likewise be produced with any solid particulatemedicament aerosol generator, by techniques known in the pharmaceuticalart.

Alternatively, one can administer the compound in a local rather thansystemic manner, for example, in a depot or sustained-releaseformulation.

Further, the present invention provides liposomal formulations of thecompounds disclosed herein and salts thereof. The technology for formingliposomal suspensions is well known in the art. When the compound orsalt thereof is an aqueous-soluble salt, using conventional liposometechnology, the same can be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed can be of anyconventional composition and can either contain cholesterol or can becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced can be reduced insize, as through the use of standard sonication and homogenizationtechniques.

The liposomal formulations containing the compounds disclosed herein orsalts thereof, can be lyophilized to produce a lyophilizate which can bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

In the case of water-insoluble compounds, a pharmaceutical compositioncan be prepared containing the water-insoluble compound, such as forexample, in an aqueous base emulsion. In such an instance, thecomposition will contain a sufficient amount of pharmaceuticallyacceptable emulsifying agent to emulsify the desired amount of thecompound. Particularly useful emulsifying agents include phosphatidylcholines and lecithin.

In particular embodiments, the compound is administered to the subjectin a therapeutically effective amount, as that term is defined above.Dosages of pharmaceutically active compounds can be determined bymethods known in the art, see. e.g., Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.). The therapeutically effectivedosage of any specific compound will vary somewhat from compound tocompound, and patient to patient, and will depend upon the condition ofthe patient and the route of delivery. As a general proposition, adosage from about 0.001 to about 50 mg/kg will have therapeuticefficacy, with all weights being calculated based upon the weight of thecompound, including the cases where a salt is employed. Toxicityconcerns at the higher level can restrict intravenous dosages to a lowerlevel such as up to about 10 mg/kg, with all weights being calculatedbased upon the weight of the compound, including the cases where a saltis employed. A dosage from about 10 mg/kg to about 50 mg/kg can beemployed for oral administration. Typically, a dosage from about 0.5mg/kg to 5 mg/kg can be employed for intramuscular injection. Particulardosages are about 1 μmol/kg to 50 μmol/kg, and more particularly toabout 22 μmol/kg and to 33 μmol/kg of the compound for intravenous ororal administration, respectively.

In particular embodiments of the invention, more than one administration(e.g., two, three, four, or more administrations) can be employed over avariety of time intervals (e.g., hourly, daily, weekly, monthly, etc.)to achieve therapeutic effects.

The present invention finds use in veterinary and medical applications.Suitable subjects include both avians and mammals, with mammals beingpreferred. The term “avian” as used herein includes, but is not limitedto, chickens, ducks, geese, quail, turkeys, and pheasants. The term“mammal” as used herein includes, but is not limited to, humans,bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.Human subjects include neonates, infants, juveniles, and adults. Inother embodiments, the subject is an animal model of cancer. In certainembodiments, the subject has or is at risk for cancer.

The following examples are not intended to limit the scope of the claimsto the invention, but are rather intended to be exemplary of certainembodiments. Any variations in the exemplified methods that occur to theskilled artisan are intended to fall within the scope of the presentinvention. As will be understood by one skilled in the art, there areseveral embodiments and elements for each aspect of the claimedinvention, and all combinations of different elements are herebyanticipated, so the specific combinations exemplified herein are not tobe construed as limitations in the scope of the invention as claimed. Ifspecific elements are removed or added to the group of elementsavailable in a combination, then the group of elements is to beconstrued as having incorporated such a change.

Example 1 Mutant KRAS-Specific siRNAs

Methods: Novel mutant-specific siRNAs (MS siRNAs) were designed based onprevious literature that suggests a 3-mismatch tolerance threshold for19-nucleotide siRNA efficacy (Naito et al., Nucleic Acids Res. 32:W124(2004)). With two or fewer mismatches between the sequence and thetarget gene, the siRNA is able to successfully bind and knock-downexpression of the gene of interest; however, at and above the 3 mismatchthreshold, the siRNA fails to recognize the target, thus allowing forexpression of the encoded protein. Custom MS siRNAs were generated usingopen source softwares provided by Sigma Aldrich, Life Technologies andDharmacon to be antisense to an artificial, hyper-mutated version of theWT KRAS gene which never actually occurs in nature, with exactly 3 pointmutations corresponding to each of the most commonly occurring KRASmutants (G12C, G12D or G12V, and G13D). Thirty flanking nucleotides wereincluded upstream and downstream of these sites in the artificial,hyper-mutated mRNA input (FIG. 1). Of note, siRNA sequences weredesigned to target two different artificial mRNA sequences; one thatsimultaneously contained specific missense mutations in codons 12 (G12Cand G12D) and 13 (G13D), and another that simultaneously containedspecific missense mutations in codons 12 (G12C and G12V) and 13 (G13D)(FIG. 1). The resultant sequence is thus antisense with 3 mismatcherrors to WT KRAS but only 2 mismatch errors to each of the 3 mutantKRAS alleles. Consequently, it was hypothesized that these MS siRNAswill optimize the task of targeting mutant KRAS while sparing the WTKRAS allele since the sequence is below the 3 mismatch threshold for theformer and above for the latter. In addition, by introducing onemutation from each of 3 different prevalent KRAS mutants in the customsequence design, the resultant siRNA has the added potential benefit ofsimultaneously targeting several KRAS mutants rather than one.

Constructs containing the WT, G12C, G12D, G12V. or G13D KRAS geneinserted into pBABE-puro retroviral expression vectors were prepared. Toexpand the vector constructs, plasmids were added to high efficiencycompetent E. coli cells and incubated in S.O.C. media on a shaker at 37°C. Cells were then plated on ampicillin agarose plates and incubated at37° C. overnight. Liquid cultures were prepared by picking and placingbacterial colonies from the overnight plates into LB broth with 1 μl/mlcarbocyclin and incubating overnight on a shaker at 37° C. Liquidcultures were performed in triplicate. Plasmid DNA from the resultantturbid cultures was purified using a QIAprep Spin Miniprep Kit (Qiagen).Successful plasmid expansion was confirmed via restriction enzyme digestusing BamHI and HindIII and gel electrophoresis. Undigested plasmidsfrom the Miniprep were further expanded in LB Broth with 0.1 μl/milcarbocyclin and then purified using a QIAprep Spin Maxiprep Kit(Qiagen).

To produce retrovirus containing the pBABE-puro-KRAS plasmids, 9×10⁶ HEK293T cells were seeded onto 6 cm cell culture plates in 293T media (DMEMwith 10% FBS and 1% penicillin-streptomycin) and incubated at 37° C., 5%CO₂. After 24 hours, plasmid DNA mixtures were prepared for eachpBABE-puro-KRAS plasmid by creating a mixture of 0.01 μg/μl plasmidconstruct and 0.01 μg/μl PCL10A pack vector plasmid to OptiMEM media. Inaddition, a L2K mixture was prepared by adding 0.05 μg/μl Lipofectamine2000 (Thermo Fisher Scientific) in OptiMEM media and incubating at roomtemperature for 5 minutes. The L2K mixture was then combined 1:1 witheach plasmid DNA mixture and incubated at room temperature for 20minutes. Media was removed from the incubated cells, then 2 ml of 293Tmedia was added to each well along with 250 μl of the plasmid DNA/L2Kmixture. After another 24 hours, the plasmid DNA/L2K media was replacedwith 293T media. After another 24 hours, the resultant viral media wascollected from the wells and replaced with fresh 293T media. Virus mediawas stored overnight on ice at 4° C. After another 24 hours, media wascollected again from the wells and added to the previously stored virus.The mixture was centrifuged at room temperature, then the supernatantwas collected. This process was repeated using mCherry and 293T mediainstead of plasmid construct to produce mCherry and empty vector virus,respectively.

To infect cells with KRAS plasmid, NIH 3T3 cells were harvested andseeded at 100,000 per well in 6 well plates in complete media (DMEM with10% Colorado calf serum and 1% pen/strep). After 4-6 hours and once thecells attached, the media was aspirated and 2 ml of complete media with10 μl/ml polybrene along with 250 μl of viral media (WT, G12C, G12D,G12V, G13D, mCherry, and empty vector) or complete media (negativecontrols) were added to each well. Cells were centrifuged at 1500 g for60 minutes at 30° C., then incubated overnight at 37° C. After 24 hours,wells were aspirated and complete media was added to each well. Afteranother 24 hours, wells were aspirated and complete media with 2 μg/mlpuromycin was added. Media was replaced with puromycin media every 24hours until no living cells remained in the negative control wells.Successful infection was further verified using mCherry expression.

RNAi knockdown was induced in KRAS-infected NIH 3T3 cells plated at adensity of 60,000 cells per 500 μl complete media per well in 24 wellplates. Sequences for the scrambled control siRNA, as well as positivecontrols (Seq #2 and #3, G12C and G12D siRNAs) previously found topotently silence wild-type and mutant KRAS were used as indicated inFIG. 1 (Fleming et al., Mol. Cancer Res. 3:413 (2005); Pecot et al.,Mol. Cancer Ther. 13:2876 (2014)). Cells were incubated in 5:1 mixtureof complete media and serum-free media, along with 20 nM siRNA (SigmaAldrich) and Lipofectamine(R) RNAiMAX transfection reagent (ThermoFisher Scientific, 2:1 ratio of transfection reagent to siRNA by volume)for 5 hours at 37° C. in 5% CO₂. Media was removed, and cells wereincubated in complete media only for another 19 hours before RNA wascollected and purified using a QIAprep Spin Miniprep Kit.

Purified RNA from siRNA treatments was quantified using aspectrophotometer, then reverse transcribed to cDNA using an iScript™cDNA Synthesis Kit (Bio-Rad). To quantify relative expression levels ofKRAS, RT-PCR reactions were performed by monitoring real-time changes influorescent intensity of SYBR green on the StepOnePlus™ Real-Time PCRSystem (Thermo Fisher Scientific). Each sample was run in triplereplicate. The StepOnePlus™ was also used to obtain RQ values using theΔΔCT analysis to calculate ΔCt values by comparing cycle threshold (Ctvalues) of KRAS to those of the target reference gene. 18s, then compareΔCt values for each siRNA to those of the NC siRNA. Error bars represent1 standard deviation. Data represents the result of one trial of twobiological replicates for WT and G12D and two trials of two biologicalreplicates for G12C, G12V, and G13D. Reverse transfection experimentswere performed in duplicate for each siRNA for each cell line.

Results: To test the efficacy of mutant-specific KRAS silencing invitro, a panel of candidate MS KRAS siRNA sequences was tested for theirability to knock-down KRAS expression in both WT and target mutantKRAS-expressing cells. The 12CD13D_1 sequence was observed to exhibit asparing of WT KRAS expression (only 4% knock-down compared to negativecontrol siRNA) while knocking down G12C (50%), G12D (82%), and G13Dmutant KRAS (66%) (FIG. 2A). The sequence was also unexpectedly noted toknock down G12V expression (58%). Additionally, 12CD13D_2 and 12CD13D_4were also found to exhibit WT sparing and mutant knockdown. However, theformer appeared to exhibit less WT sparing than 12CD13D_1 and the latterless KRAS knockdown in all cell lines (FIG. 2A). The remaining sequencesexhibited either low potency against mutant KRAS, high KRAS knockdown inthe WT KRAS cell line, or both (FIG. 2A).

In addition, G12C- and G12D-specific siRNA sequences (Fleming et al.,Mol. Cancer Res. 3:413 (2005)) were tested in order to compare theefficacy of MS siRNA sequences against those previously demonstrated toexhibit target-specificity. However, the mutant-specificity of thesesequences was not confirmed, and the sequences did not exhibit theexpected preferential knockdown of G12C and G12D mutant KRAS,respectively, over the WT or other mutant alleles (FIG. 2B).

KRAS siRNA sequences 12CD13D_1 and 12CD13D_4 were tested in a KRAS G12Dmutant lung cancer cell line. Using a control siRNA (Scr) and twopreviously validated KRAS siRNAs (Seq #2 and #3), it was demonstratedthat customized, mutant specific KRAS siRNA sequences 12CD13D_1 and12CD13D_4 are highly effective at silencing KRAS protein expression(FIG. 3).

Testing all possible siRNA sequence permutations between our customsiRNA sequences. In order to verify the best possible custom KRAS siRNAsequences (leading sequences being 12CD13D_1 and 12CD3D_4), a library ofsequences (12CD13D_A thru 12CD13D_F) were tested that incrementally movedownstream between 12CD13D_1 and 12CD13D_4 (FIG. 4).

Following stable transduction of 3T3 cells with either wild-type (WT) ormutant G12C, G12D, G12V and G13D human KRAS sequences, the cells weretransfected with KRAS siRNA sequences listed in FIG. 5. Twenty-fourhours after transfection, cells were lysed, RNA collected and cDNA wasmade. Quantitative qPCR was performed for KRAS using 18s as ahouse-keeping gene. It was found that the leading 12CD13D_1 and12CD13D_4 custom sequences were still the best overall at silencingmutant KRAS, while the other possible KRAS siRNA sequences (“A” thru“F”) were less potent overall at silencing the different KRAS mRNAsequences. On this experiment the custom KRAS siRNA 12CD13D_4 sequencewas best at sparing the WV sequence.

Discussion: Although numerous efforts have been made to target mutantKRAS, no direct inhibitors are currently in clinical use. Moreover, mostcurrent small-molecule cancer therapeutics exhibit low targetspecificity, resulting in adverse toxicity in non-cancerous cells (Pecotet al., Nat. Rev. Cancer 11:59 (2011)). Despite current headways ininhibiting downstream effectors in the KRAS signaling pathway, KRASremains an elusive target for drug development (Cox et al., Nat. Rev.Drug Discov. 13:828 (2014)). As such, this study investigated theefficacy of novel MS siRNA as a means of selectively inhibitingexpression of mutant KRAS.

Based on these preliminary findings, the 12CD13D_1 and 12CD13D_2 siRNAsequences were chosen as lead candidates for further investigation astherapeutic agents because of their high mutant-specificity and potency.To a lesser extent, 12CD13D_4 is also a promising candidate; however,its low efficiency in knocking down KRAS expression in mutant targetssuggests that it may not be effective as a clinically relevanttherapeutic agent. By contrast, the G12C- and G12D-specific siRNA do notappear to exhibit any sort of sparing of the WT KRAS allele.

In addition, the low specificity of both sequences designed to targetthe G12V rather than the G12D point mutation suggests a greatertolerance for WT KRAS in G12V-targeting sequences.

These preliminary findings collectively suggest the viability of novelMS siRNAs as a mutant-specific vehicle for silencing oncogenic KRAS.With its potential as an effective payload with mutant KRAS specificity,novel mutant-specific siRNAs present a promising avenue of pursuit fordrugging the formerly “undruggable.”

Example 2 Antisense Oligonucleotides Targeted to Synthetic Mutant KRAS

A series of antisense oligonucleotide (ASO) sequences were created thattarget a mutant KRAS of the invention comprising the mutations G12C,G12D and G13D (SEQ ID NO:53) relative to the wild-type KRAS sequence(SEQ ID NO:52) (FIG. 6 and Table 2). In FIG. 6, the black bars representnucleotides that have a 2′-methoxy-ethyl (MOE) modification, while thegray regions represent the “Gapmer” that is composed of DNA. Theschematic is not to scale and the black bars are composed of 5 flankingMOE-modified nucleotides on each side while there is a 10 nucleotideGapmer. The ASOs incorporate phosphorothioate linkages (PS, designatedby a “*”) between all nucleotides, a 10-nt Gapmer (underlined), and 5flanking 2′-MOE (methoxy-ethyl) modifications (bold). The ASOs weretested to identify single-stranded RNA and/or DNA sequences thatmaintain the ability to silence several KRAS mutations.

TABLE 2 ASO sequences SEQ ASO ID Number Sequence NO  1T*C*A*T*A*A*G*C*T*C*C*A*A*C*T* A*C*C*A*C 54  2G*T*C*A*T*A*A*G*C*T*C*C*A*A*C* T*A*C*C*A 55  3C*G*T*C*A*T*A*A*G*C*T*C*C*A*A* C*T*A*C*C 56  4A*C*G*T*C*A*T*A*A*G*C*T*C*C*A* A*C*T*A*C 57  5T*A*C*G*T*C*A*T*A*A*G*C*T*C*C* A*A*C*T*A 58  6C*T*A*C*G*T*C*A*T*A*A*G*C*T*C* C*A*A*C*T 59  7C*C*T*A*C*G*T*C*A*T*A*A*G*C*T*C*C*A*A*C 60  8G*C*C*T*A*C*G*T*C*A*T*A*A*G*C*T*C*C*A*A 61  9T*G*C*C*T*A*C*G*T*C*A*T*A*A*G* C*T*C*C*A 62 10T*T*G*C*C*T*A*C*G*T*C*A*T*A*A* G*C*T*C*C 63 11C*T*T*G*C*C*T*A*C*G*T*C*A*T*A* A*G*C*T*C 64 12T*C*T*T*G*C*C*T*A*C*G*T*C*A*T* A*A*G*C*T 65 13C*T*C*T*T*G*C*C*T*A*C*G*T*C*A* T*A*A*G*C 66 14A*C*T*C*T*T*G*C*C*T*A*C*G*T*C* A*T*A*A*G 67 15C*A*C*T*C*T*T*G*C*C*T*A*C*G*T* C*A*T*A*A 68 16G*C*A*C*T*C*T*T*G*C*C*T*A*C*G* T*C*A*T*A 69

Four separate A431 cell lines were engineered to remove expression ofwild-type KRAS and express one of the mutations G12C, G12D, G13D, orG12V. Each cell line was treated with free ASOs at 1.1 μM for 48 hours,and qPCR was run. The results are shown in FIG. 7. While several of theASOs were demonstrated to silence one or more of the mutations, it wasfound that ASO15 and ASO16 were very potent at silencing all 4 of themost common KRAS mutations.

Some of the top hits were re-evaluated, and again ASO15 and ASO16 werefound to potently silence all 4 of the KRAS mutations in adose-responsive manner (FIG. 8). Cells were treated with free ASOs at1.1 μM and 10 μM for 48 hours, then qPCR was run for mutant KRAS.

Example 3 Modified ASO16 Sequences

Starting with the potent ASO16, modified versions were prepared toidentify ASOs with increased mutation specificity by targeting the ASOto mutations that exist in nature, e.g., single mutations (G12C, G12D,G12V, or G13D). Modifications included base substitutions and the use oflocked nucleic acids. The modified sequences are shown in Table 3. TheASOs incorporates phosphorothioate linkages (PS, designated by a “*”)between all nucleotides, a 10-nt Gapmer (underlined), and 5 flanking2′-MOE (methoxy-ethyl) modifications (bold). The last four sequences usea single locked nucleic acid (LNA, denoted by “+” before the base) inplace of an MOE to increase melting temperatures at specific sites. TheLNA consists of a methylene bridge connecting the 2′ and 4′ carbons.

TABLE 3 Modified ASO16 sequences ASO Number Sequence SEQ ID NO 16G*C*A*C*T*C*T*T*G*C*C*T*A*C*G* T*C*A*T*A 69 ASO16-G12C G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*C*A 70 ASO16-G12DG*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*T*C 71 ASO16-G12VG*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*A*C 77 ASO16-G13DG*C*A*C*T*C*T*T*G*C*C*T*A*C*G*T*C*A*C*C 73 ASO16- G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*C*+A 74 G12C-LNA ASO16-G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*+T*C 75 G12D-LNA ASO16-G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*+A*C 76 G12V-LNA ASO16-G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*+T*C*A*C*C 77 G13D-LNA

The ASOs were tested on the A431 cell lines as described above. Theresults are shown in FIG. 9. Improved potency for each KRAS mutation wasfound for: 1) G12C using ASO16-G12C and ASO-G12C-LNA, 2) G12D usingASO16-G12D and ASO16-G12D-LNA, 3) G12V using ASO16-G12V andASO16-G12V-LNA and 4) G13D using ASO16-G13D and ASO16-G13D-LNA. Theseresults indicate that the increased mutation specificity of these ASOsequences led to increased potency.

Example 4 Fully Modified siRNA Sequences

Several of the ASOs targeted to single KRAS mutations described inExample 3 were converted to siRNA molecules in an effort to identifysequences that maximize the reduction of expression of mutated sequenceswhile sparing the wild-type KRAS sequence.

These siRNAs were then fully modified (FM) to minimize nucleasedegradation and immune stimulation. While these types of modificationsoften attenuate the silencing activity of siRNAs, the inventors havedeveloped several fully modified siRNA sequences that retain all ornearly all of their silencing activity compared to unmodified siRNAs.

Table 4 lists the fully modified siRNA sequences that were prepared.

Each of the siRNAs was tested in A431 cells engineered to either expressWT or the targeted KRAS mutation. For mutant expressing A431 cells, theWT allele was deleted via CRISPR, so the expression shown is onlyreflective of the mutant mRNA. Cells were transfected with a low dose(20 nM) of negative control (NC) or the FM KRAS siRNAs and qPCR for KRASexpression was run on RNA isolated 24 hours later.

For siRNAs targeting the G12C mutation, it was found that D1-G12C-FM and12-G12C-FM were both able to reduce mutant KRAS G12C while spare thewild-type expression (FIG. 10). Notably, not all of the siRNAs wereequivalent in suppressing G12C KRAS or sparing wild-type KRAS. As shownin FIG. 11, it was found that some (hatched bars) mutant-specific (MS)iterations of the ‘parent’ D1, D2, and D4 sequences were more potent(panel 1). It was found that several of the MS sequences more potentlyreduced the mutant over WT sequences (see D1-G12C, D2-G12C, D4-G12C),unlike a pan-KRAS Seq 2 (panel 2). Finally, it was found that FMiterations of these MS sequences showed retained silencing activity onmutant KRAS over WT KRAS expression (e.g., D2-G12C-FM-F) (panel 3).

TABLE 4 Fully modified siRNA sequences Name Strand Sequence (5′-3′)SEQ ID NO D1-G12C-FM S[mG]*[mA]*[mG][mC][mU][mU][2flG][mU][2flG][2flG][2flC][mG][mU][mA][mG][mG][mC][mA][mA][mG][mA]78 AS[mU]*[2flU]*[mG][mC][mC][2flU][mA][mC][mG][mC][mC][mA][mC][2flA][mA][2flG][mC][mU][mC]*[mC]*[mA]79 D1-G12D-FM S[mG]*[mA]*[mG][mC][mU][mG][2flA][mU][2flG][2flG][2flC][mG][mU][mA][mG][mG][mC][mA][mA][mG][mA]80 AS[mU]*[2flU]*[mG][mC][mC][2flU][mA][mC][mG][mC][mC][mA][mU][2flC][mA][2flG][mC][mU][mC]*[mC]*[mA]81 D1-G13D-FM S[mG]*[mA]*[mG][mC][mU][mG][2flG][mU][2flG][2flA][2flC][mG][mU][mA][mG][mG][mC][mA][mA][mG][mA]82 AS[mU]*[2flU]*[mG][mC][mC][2flU][mA][mC][mG][mU][mC][mA][mU][2flC][mA][2flG][mC][mU][mC]*[mC]*[mA]83 D2-G12C-FM S[mA]*[mG]*[mU][mU][mG][mG][2flA][mG][2flC][2flU][2flU][mG][mU][mG][mG][mC][mG][mU][mA][mG][mG]84 AS[mU]*[2flA]*[mC][mG][mC][2flC][mA][mC][mA][mA][mG][mC][mU][2flC][mC][2flA][mA][mC][mU]*[mA]*[mC]85 D2-G12D-FM S[mA]*[mG]*[mU][mU][mG][mG][2flA][mG][2flC][2flU][2flG][mA][mU][mG][mG][mC][mG][mU][mA][mG][mG]86 AS[mU]*[2flA]*[mC][mG][mC][2flC][mA][mU][mC][mA][mG][mC][mU][2flC][mC][2flA][mA][mC][mU]*[mA]*[mC]87 D2-G13D-FM S[mA]*[mG]*[mU][mU][mG][mG][2flA][mG][2flC][2flU][2flG][mG][mU][mG][mA][mC][mG][mU][mA][mG][mG]88 AS[mU]*[2flA]*[mC][mG][mU][2flC][mA][mC][mC][mA][mG][mC][mU][2flC][mC][2flA][mA][mC][mU]*[mA]*[mC]89 D4-G13D-FM S[mG]*[mG]*[mU][mA][mG][mU][2flU][mG][2flG][2flA][2flG][mC][mU][mG][mG][mU][mG][mA][mC][mG][mU]90 AS[mG]*[2flU]*[mC][mA][mC][2flC][mA][mG][mC][mU][mC][mC][mA][2flA][mC][2flU][mA][mC][mC]*[mA]*[mC]91 D1-G12V-FM S[mA]*[mG]*[mU][mU][mG][mG][2flA][mG][2flC][2flU][2flG][mU][mU][mG][mG][mC][mG][mU][mA][mG][mG]92 AS[mG]*[2flU]*[mC][mA][mC][2flC][mA][mG][mC][mU][mC][mC][mA][2flA][mC][2flU][mA][mC][mC]*[mA]*[mC]96 D2-G12V-FM S[mG]*[mA]*[mG][mC][mU][mG][2flU][mU][2flG][2flG][2flC][mG][mU][mA][mG][mG][mC][mA][mA][mG][mA]94 AS[mU]*[2flU]*[mG][mC][mC][2flU][mA][mC][mG][mC][mC][mA][mA][2flC][mA][2flG][mC][mU][mC]*[mC]*[mA]95 D1-G12D-FM-F S[mG]*[mA]*[mG][mC][mU][mG][2flA][mU][2flG][2flG][2flC][mG][mU][mA][mG][mG][mC][mA][mA][mG][mA]96 AS[mU]*[2flU]*[mG][mC][mC][2flU][mA][mC][mG][mC][mC][mA][2flU][2flC][mA][2flG][mC][mU][mC]*[mC]*[mA]97 D1-G13D-FM-F S[mG]*[mA]*[mG][mC][mU][mG][2flG][mU][2flG][2flA][2flC][mG][mU][mA][mG][mG][mC][mA][mA][mG][mA]98 AS[mU]*[2flU]*[mG][mC][mC][2flU][mA][mC][mG][2flU][mC][mA][mC][2flC][mA][2flG][mC][mU][mC]*[mC]*[mA]99 D2-G12C-DM-F S[mA]*[mG]*[mU][mU][mG][mG][2flA][mG][2flC][2flU][2flU][mG][mU][mG][mG][mC][mG][mU][mA][mG][mG]100 AS[mU]*[2flA]*[mC][mG][mC][2flC][mA][mC][2flA][mA][mG][mC][mU][2flC][mC][2flA][mA][mC][mU]*[mA]*[mC]101 D2-G12D-FM-F S[mA]*[mG]*[mU][mU][mG][mG][2flA][mG][2flC][2flU][2flG][mA][mU][mG][mG][mC][mG][mU][mA][mG][mG]102 AS[mU]*[2flA]*[mC][mG][mC][2flC][mA][2flU][mC][mA][mG][mC][mU][2flC][mC][2flA][mA][mC][mU]*[mA]*[mC]103 D2-G13D-FM-F S[mA]*[mG]*[mU][mU][mG][mG][2flA][mG][2flC][2flU][2flG][mG][mU][mG][mA][mC][mG][mU][mA][mG][mG]104 AS[mU]*[2flA]*[mC][mG][2flU][2flC][mA][mC][mC][mA][mG][mC][mU][2flC][mC][2flA][mA][mC][mU]*[mA]*[mC]105 D1-G12V-FM-F S[mA]*[mG]*[mU][mU][mG][mG][2flA][mG][2flC][2flU][2flG][mG][mU][mG][mA][mC][mG][mU][mA][mG][mG]106 AS[mU]*[2flA]*[mC][mG][mC][2flC][mA][2flA][mC][mA][mG][mC][mU][2flC][mC][2flA][mA][mC][mU]*[mA]*[mC]107 D2-G12V-FM-F S[mG]*[mA]*[mG][mC][mU][mG][2flU][mU][2flG][2flG][2flC][mG][mU][mA][mG][mG][mC][mA][mA][mG][mA]108 AS[mU]*[2flU]*[mG][mC][mC][2flU][mA][mC][mG][mC][mC][mA][2flA][2flC][mA][2flG][mC][mA][mC]*[mC]*[mA]109 Seq2-FM S[mG]*[mU]*[mC][mU][mC][mU][2flU][mG][2flG][2flA][2flU][mA][mU][mU][mC][mU][mC][mG][mA]110 AS[mU]*[2flC]*[mG][mA][mG][2flA][mA][mU][mA][mU][mC][mC][mA][2flA][mG][2flA][mG][mA][mC]*[mA]*[mG]111 Seq3-FM S[mC]*[mA]*[mG][mC][mU][mA][2flA][mU][2flU][2flC][2flA][mG][mA][mA][mU][mC][mA][mU][mU]112 AS[mA]*[2flA]*[mU][mG][mA][2flU][mU][mC][mU][mG][mA][mA][mU][2flU][mA][2flG][mC][mU][mG]*[mU]*[mA]113 S-sense strand AS-antisense strand m-2′-O-methyl on sugar moieties2fl-2′-fluoro on sugar moieties *-phosphorothioate in betweennucleotides

For siRNAs targeting the G12D mutation, it was found that D1-G12D-FM,D2-G12D-FM and D2-G12D-FM-F were all able to reduce mutant KRAS G12D(FIG. 12). D1-G12D-FM and D2-G12D-FM were also able to spare WTexpression (FIG. 12). Notably, not all of the siRNAs were equivalent insuppressing G12D KRAS or sparing wild-type KRAS. As shown in FIG. 13, itwas found that some (hatched bars) mutant-specific (MS) iterations ofthe ‘parent’ D1, D2 and D4 sequences were more potent (panel 1). It wasfound that several of the MS sequences more potently reduced the mutantover WT sequences (see D1-G12D), unlike a pan-KRAS Seq 2 (panel 2).Finally, it was found that FM iterations of these MS sequences showedretained silencing activity on mutant KRAS over WT KRAS expression(e.g., D1-G12D-FM and D2-G12D-FM) (panel 3).

For siRNAs targeting the G12V mutation, it was found that V1-G12V-FM andV2-G12D-FM were able to reduce mutant KRAS G12V while also able to spareWT expression (FIG. 14). Notably, not all of the siRNAs were equivalentin suppressing G12D KRAS or sparing wild-type KRAS. As shown in FIG. 15,it was found that some (hatched bars) mutant-specific (MS) iterations ofthe ‘parent’ D1, D2 and D4 sequences were more potent (panel 1). It wasfound that several of the MS sequences more potently reduced the mutantover WT sequences (see D2-G12V), unlike a pan-KRAS Seq2 (panel 2).Finally, it was found that FM iterations of these MS sequences showedretained silencing activity on mutant KRAS over WT KRAS expression(e.g., D1-G12V-FM and D2-G12V-FM) (panel 3).

For siRNAs targeting the G13D mutation, it was found that D2-G13D-FM andD2-G13D-FM-F were able to reduce mutant KRAS G12D (FIG. 16).D2-G12D-FM-F was also able to spare WT expression (FIG. 16). Notably,not all of the siRNAs were equivalent in suppressing G12D KRAS orsparing wild-type KRAS. As shown in FIG. 17, it was found that some(hatched bars) mutant-specific (MS) iterations of the ‘parent’ D1, D2and D4 sequences were more potent (panel 1). It was found that severalof the MS sequences more potently reduced the mutant over WT sequences(see D1-G13D and D4-G13D), unlike a pan-KRAS Seq 2 (panel 2). Finally,it was found that FM iterations of these MS sequences showed retainedsilencing activity on mutant KRAS over WT KRAS expression (e.g.,D2-G13D-FM and D2-G13D-FM-F) (panel 3).

Example 5 Additional Fully Modified siRNA Sequences

siRNAs comprising the sequence of SEQ ID NO:50 or SEQ ID NO:51 were usedas positive controls in the experiments described in Example 1. Fullymodified versions of these siRNAs were prepared to test theirspecificity and potency. The sequences of the FM siRNAs are shown inTable 4. The HCT116 (colon cancer, KRAS G13D) and LU65 (lung cancer,KRAS G12C) cell lines were transfected with 20 nM of either unmodifiedSeq2 or Seq3, or fully chemically modified (FM) Seq2-FM or Seq3-FM siRNAsequences. Both Seq2-FM and Seq3-FM surprisingly maintained fullsilencing of KRAS activity, especially at 48 hours post-treatment (FIG.18).

All publications, patents, and patent applications are hereinincorporated by reference to the same extent as if each individualpublication, patent, or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the list of the foregoingembodiments and the appended claims.

1. An antisense oligonucleotide targeted to a synthetic human KRAS mRNAthat encodes the missense mutations G12C, G12D, and G13D, wherein theantisense oligonucleotide is 16-25 nucleotides in length and comprisesthe sequence (SEQ ID NO: 114) TCTTGCCTACGTCATA.


2. (canceled)
 3. The antisense oligonucleotide of claim 1, consisting ofthe sequence CACTCTTGCCTACGTCATAA (SEQ ID NO:115) orGCACTCTTGCCTACGTCATA (SEO ID NO: 116).
 4. (canceled)
 5. The antisenseoligonucleotide of claim 1, wherein the antisense oligonucleotidecomprises at least one phosphorothioate linkage, optionally allphosphorothioate linkages.
 6. (canceled)
 7. The antisenseoligonucleotide of claim 1, wherein the antisense oligonucleotidecomprises at least one modified nucleotide at or near the 5′ end and/orthe 3′ end, optionally at least three modified nucleotides at each ofthe 5′ end and the 3′ end.
 8. (canceled)
 9. The antisenseoligonucleotide of claim 7, wherein the modified nucleotide is a2′-O-methoxyethyl (2′-MOE)-modified nucleotide.
 10. The antisenseoligonucleotide of claim 9, wherein the antisense oligonucleotidecomprises at least three 2′-MOE-modified nucleotides at each of the 5′end and/or the 3′ end.
 11. The antisense oligonucleotide of claim 10,wherein the antisense oligonucleotide consists of a sequence selectedfrom: a) (SEQ ID NO: 68) C*A*C*T*C*T*T*G*C*C*T*A*C*G*T*C*A*T*A*A; or b)(SEQ ID NO: 69) G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*T*C*A*T*A;

wherein * indicates a phosphorothioate linkage and bold indicates a2′-MOE-modified nucleotide.
 12. The antisense oligonucleotide of claim7, wherein at least one modified nucleotide is a locked nucleic acid.13. An antisense oligonucleotide targeted to a naturally-occurring humanKRAS mRNA encoding a mutation selected from G12C, G12D, G12V, and G13D,wherein the antisense oligonucleotide is 16-25 nucleotides in length andcomprises a sequence selected from: a) TCTTGCCTACGCCACA (SEQ ID NO:117)targeted to a human KRAS mRNA encoding a G12C mutation; b)TCTTGCCTACGCCATC (SEQ ID NO:118) targeted to a human KRAS mRNA encodinga G12D mutation; c) TCTTGCCTACGCCAAC (SEQ ID NO:119) targeted to a humanKRAS mRNA encoding a G12V mutation; d) TCTTGCCTACGTCACC (SEQ ID NO:120)targeted to a human KRAS mRNA encoding a G13D mutation; or e) a sequenceat least 90% identical to any one of a) to d); wherein the antisenseoligonucleotide comprises at least one non-naturally occurring chemicalmodification.
 14. (canceled)
 15. The antisense oligonucleotide of claim13, wherein the antisense oligonucleotide consists of a sequenceselected from: a) GCACTCTTGCCTACGCCACA (SEQ ID NO:117) targeted to ahuman KRAS mRNA encoding a G12C mutation; b) GCACTCTTGCCTACGCCATC (SEQID NO:118) targeted to a human KRAS mRNA encoding a G12D mutation; c)GCACTCTTGCCTACGCCAAC (SEQ ID NO:119) targeted to a human KRAS mRNAencoding a G12V mutation; or d) GCACTCTTGCCTACGTCACC (SEQ ID NO:120)targeted to a human KRAS mRNA encoding a G13D mutation.
 16. Theantisense oligonucleotide of claim 13, wherein the antisenseoligonucleotide comprises at least one phosphorothioate linkage,optionally all phosphorothioate linkages.
 17. (canceled)
 18. Theantisense oligonucleotide of claim 13, wherein the antisenseoligonucleotide comprises at least one modified nucleotide at or nearthe 5′ end and/or the 3′ end, optionally at least the modifiednucleotides at each of the 5′ end and the 3′ end.
 19. (canceled)
 20. Theantisense oligonucleotide of claim 18, wherein the modified nucleotideis a 2′-MOE-modified nucleotide.
 21. The antisense oligonucleotide ofclaim 20, wherein the antisense oligonucleotide comprises at least three2′-MOE-modified nucleotides at each of the 5′ end and/or the 3′ end. 22.The antisense oligonucleotide of claim 21, wherein the antisenseoligonucleotide consists of a sequence selected from: a) (SEQ ID NO: 70)G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*C*A; b) (SEQ ID NO: 71)G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*T*C; c) (SEQ ID NO: 72)G*C*A*C*T*C*T*T*G*C*C*T*A*C'G*C*C*A*A*C; or d) (SEQ ID NO: 73)G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*T*C*A*C*C;

wherein * indicates a phosphorothioate linkage and bold indicates a2′-MOE-modified nucleotide.
 23. The antisense oligonucleotide of claim18, wherein at least one modified nucleotide is a locked nucleic acid.24. The antisense oligonucleotide of claim 23, wherein the antisenseoligonucleotide consists of a sequence selected from: a) (SEQ ID NO: 74)G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*C*+A; b) (SEQ ID NO: 75)G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*+T*C; c) (SEQ ID NO: 76)G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*C*C*A*+A*C; or d) (SEQ ID NO: 77)G*C*A*C*T*C*T*T*G*C*C*T*A*C*G*+T*C*A*C*C;

wherein * indicates a phosphorothioate linkage, bold indicates a2′-methoxymethyl-modified nucleotide, and + indicates the followingnucleotide is a locked nucleic acid.
 25. (canceled)
 26. A nucleic acidmolecule encoding the antisense oligonucleotide of claim
 13. 27-38.(canceled)
 39. A composition comprising the antisense oligonucleotide ofclaim
 13. 40. A composition comprising two or more of the antisenseoligonucleotide of claim 13, in any combination, wherein the two or moreantisense oligonucleotides each comprise a different sequence. 41-42.(canceled)
 43. A pharmaceutical composition comprising the antisenseoligonucleotide of claim 13 and a pharmaceutically acceptable carrier.44. (canceled)
 45. A method of treating cancer in a subject in needthereof, wherein the cancer comprises a mutant human KRAS genecomprising one or more of the missense mutations G12C, G12D, G12V, andG13D, the method comprising delivering to the subject the antisenseoligonucleotide of claim 13, thereby treating cancer in the subject. 46.(canceled)